Allelic Variants of Human Factor VIII

ABSTRACT

Disclosed are compositions and methods related to Factor VIII.

CROSS REFERENCE TO RELATED PATENT APPLICATIONS

This application claims priority to U.S. Provisional Application No.60/634,065 filed Dec. 6, 2004, and U.S. Provisional Application No.Unassigned, entitled “Allelic Variants of Human Factor VIII” filed onNov. 16, 2005, both of which are herein incorporated by reference intheir entireties.

I. BACKGROUND

Hemophilia is a congenital bleeding disorder. Patients with Hemophilia Ahave either absent, decreased or defective production of the bloodclotting protein, Factor VIII (FVIII). Those with Hemophilia B havesimilar problems with Factor IX (FIX). Hemophilia is characterized as“severe” when the activity of the affected clotting factor (FVIII orFIX) is less than 1% of normal. Severe Hemophilia is often associatedwith spontaneous bleeding (i.e. bleeding not caused by trauma orinjury). Hemophilia is termed “mild” when the relevant clotting factoractivity is 6-24% of normal. Hemophilia is referred to as “moderate”when clotting factor activity is between 1% and 5% of normal.Approximately 70% of Hemophilia patients have severe disease and canrequire treatment for bleeding several times per month.

Most patients that have Hemophilia A or B are treated by replacing theirmissing coagulation factor with FVIII or FIX that is either derived fromplasma or developed using recombinant technology. One of the mostserious complications of the treatment of Hemophilia is the developmentof ‘inhibitors’. ‘Inhibitors’ are antibodies to FVIII or FDC that candevelop in patients with Hemophilia following replacement therapy withthe Missing coagulation factor. The management of Hemophilia patientswith inhibitors is difficult. Clinically, most inhibitors are detectedwhen patients fail to respond to standard replacement therapy. Inhibitorlevels are measured in Bethesda units (BU). In general a patient havinga BU exceeding 10 is considered refractory to treatment with humanFVIII.

Inhibitors are usually first detected using a sensitive clotting-basedassay, variably referred to as an inhibitor screen or a mixing study.The coagulation factor specificity of the suspected inhibitor is nextcommonly determined by performing a set of clotting-based factoractivity assays where each is specific for one of the candidatecoagulation proteins potentially being targeted. The presence andspecificity of an inhibitor is most often confirmed by performing themore specific clotting-based test known as the Bethesda assay. Theplasma level (i.e. titer) of an inhibitor, likewise, is typicallymeasured by the Bethesda assay, and is defined in terms of Bethesdaunits (BU).

Acquired Hemophilia is a spontaneous development of inhibitors to one'sown FVIII. Acquired Hemophilia occurs in about one person per million.The underlying cause of inhibitor development in acquired hemophilia isunknown but is associated with many conditions including pregnancy,autoimmune disease, the use of certain medications or cancer AcquiredHemophilia A: A Concise Review Franchini et. al. American Journal ofHematology 80:55-63 (2005). Patients with acquired Hemophilia maypresent to the hospital as a result of a severe spontaneous bleedingepisode. These bleeding episodes are very difficult to control, and willnot typically respond to treatment with FVIII.

Treatment of Hemophilia patients (both A and B) and patients with othercoagulation factor deficiencies is normally based on replacement therapy(substitution of the missing clotting factor).

The replacement clotting factors are typically obtained from humanplasma or from recombinant (genetically engineered) preparations. Humanplasma-derived clotting factors have the inherent risk of potentiallytransmitting certain viruses. Antibodies or ‘inhibitors’ can developfollowing treatment with either human plasma factor concentrates orrecombinant clotting factor preparations. Alloantibodies react with thereplacement fVIII product but not with the patient's endogenous fVIII.Occassionally patients develop autoantibodies in addition tialloantibodies in response to infused fVIII. When this occurs a mild ormoderate patient may become a severe patient. The development ofinhibitors is very problematic as injected replacement therapy isfrequently ‘neutralized’ or made ineffective by the inhibitor shortlyafter infusion. Treatment options are available for treating Hemophiliapatients that develop inhibitors include are high dose FVIII or FIXtreatment (to overcome the inhibitor), for hemophilia A NonvoSeven(rfVIIa) porcine FVIII (FVIII derived from the plasma of pigs) orbypassing agents such as prothrombin complex concentrates (PCCs) oractivated prothrombin complex concentrates (e.g., FEIBA and other APCCs)which enhance the hemostatic process without the need of FVIII or FIX.

FVIII is also associated with other diseases and disorders, as outlinedbelow. Elevated levels of FVIII are an important risk factor for venousthrombosis and may also be associated with arterial thrombosis.Thrombosis is the formation of a clot or thrombus inside a blood vessel,obstructing the flow of blood through the circulatory system.Thromboembolism is a general term describing both thrombosis and itsmain complication: dislodgement of a clot and embolization. VonWillebrand's disease is due to an abnormality, either quantitative orqualitative, of the Von Willebrand factor (vWF), which is a largemultimeric glycoprotein that functions as the carrier protein for factorVIII (FVIII). vWF also is required for normal platelet adhesion. Assuch, vWF functions in both primary (involving platelet adhesion) andsecondary (involving FVIII) hemostasis. In primary hemostasis, vWF bindson platelets to its specific receptor glycoprotein Ib and acts as anadhesive bridge between the platelets and damaged subendothelium at thesite of vascular injury. In secondary hemostasis, vWF protects FVIIIfrom degradation and delivers it to the site of injury. What is neededin the art are methods and compositions for screening for and treatingdiseases and disorders related to FVILL and in the case of hemophilia Aless antigenic and less immunogenic vVIII replacement preparations andin the case of prothombotic fVIII improved means to neutralize coagulantactivity.

II. SUMMARY

Disclosed is a method of categorizing a haplotype in a FVIII genecomprising, amplifying regions of the FVIII gene, determining ahaplotype of the FVIII gene from DNA sequence within the amplifiedregions, and categorizing the haplotype as being an H1 (SEQ ID NO: 1),H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO: 4), H5 (SEQ ID NO:5), or H6 (SEQ ID NO: 6).

Disclosed is a method of categorizing a haplotype in a FVIII genecomprising, detecting a FVIII protein and categorizing the haplotype ofthe FVIII gene encoding the detected FVIII protein as being an H1 (SEQID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO: 4), H5(SEQ ID NO: 5), or H6 (SEQ ID NO: 6).

Disclosed is a method of reducing the generation of anti-FVIIIantibodies that inhibit or impair FVIII treatment comprising, detectinga haplotype in a FVIII gene in a subject, matching a replacement FVIIItherapy to the detected haplotype, and administering the matchedreplacement FVIII therapy to the subject. The haplotype can be H1 (SEQID NO: 1), H2′(SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO: 4), H5(SEQ ID NO: 5), or H6 (SEQ ID NO: 6).

Disclosed is a method of preventing the generation of anti-FVIIIantibodies that inhibit or impair FVIII treatment comprising, detectinga haplotype in a FVIII gene in a subject, matching a replacement FVIIItherapy to the detected haplotype, and administering the matchedreplacement FVIII therapy to the subject. The haplotype can be H1 (SEQID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO: 4), H5(SEQ ID NO: 5), or H6 (SEQ ID NO: 6).

Disclosed is a method of maximizing efficacy of transfusion therapy in asubject with a hemostatic disorder comprising, determining whether theFVIII haplotype of a subject having a hemostatic disorder is H1 (SEQ IDNO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO: 4), H5 (SEQID NO: 5), or H6 (SEQ ID NO: 6) and administering an appropriatetransfusion product to the subject based on the results. The transfusionproduct can be a recombinant FVIII. The transfusion product can beplasma derived FVIII.

Disclosed is a method of administering a blood product to a subject inneed of FVIII comprising, obtaining a haplotype in a FVIII gene of ablood product recipient, determining which type of blood product therecipient should receive, and administering to the subject in needthereof an appropriate blood product. The blood product can be pooledblood plasma derived from more than one blood donor. The blood productcan be a plasma-derived FVIII preparation. The pooled blood plasma canbe obtained by detecting a haplotype in a FVIII gene of a blood plasmadonor and placing the blood plasma of the blood plasma donor in anappropriate FVIII haplotype pool based on the results.

Disclosed is a method of blood plasma pooling comprising, detecting ahaplotype in a FVIII gene of a blood plasma donor and placing bloodplasma of the blood plasma donor in an appropriate pool based on theresults. Disclosed is a pooled blood plasma product obtained throughthis method.

Disclosed is a method of blood plasma pooling comprising, detecting ahaplotype in a FVIII gene of a whole blood donor, receiving whole bloodfrom the whole blood donor, separating plasma from the whole blood, andpooling the plasma with plasma obtained from other donors with the samewhere possible or most closely matched haplotype. Disclosed is a pooledblood plasma product obtained through this method.

Disclosed is a method of preparing a plasma-derived FVIII productcomprising, determining the haplotype of blood plasma, wherein thehaplotype is H1 (SEQ ID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4(SEQ ID NO: 4), H5 (SEQ ID NO: 5), or H6 (SEQ ID NO: 6). and preparing aplasma-derived FVIII product from the haplotyped blood plasma whereinthe resulting FVIII product is homogenous for single haplotype orenriched with selected haplotypes with respect to FWD content. Disclosedis a plasma-derived FVIII product obtained through this method.

Disclosed is a method of treating a subject with a hemostatic disordercomprising, identifying a subject with a hemostatic disorder,determining the FVIII haplotype of the subject, wherein the haplotype isH1 (SEQ ID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO:4), H5 (SEQ ID NO: 5), or H6 (SEQ ID NO: 6) and administering anappropriate FVIII gene replacement product to the subject based on theresults. The hemostatic disorder can be congenital hemophilia A. Thehemostatic disorder can be acquired hemophilia A.

Disclosed is a method of treating a subject with a hemostatic disordercomprising, identifying a subject with a hemostatic disorder,determining the FV111 haplotype of the subject, wherein the haplotype isH1 (SEQ ID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO:4), H5 (SEQ ID NO: 5), or H6 (SEQ ID NO: 6) and administering anappropriate plasma-derived FVIII product to the subject based on theresults. The hemostatic disorder can be congenital hemophilia A. Thehemostatic disorder can be acquired hemophilia A.

Disclosed is a method of treating a subject with a hemostatic disordercomprising, identifying a subject with a hemostatic disorder,determining the FVIII haplotype of the subject, wherein the haplotype isH1 (SEQ ID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO:4), H5 (SEQ ID NO: 5), or H6 (SEQ ID NO: 6) and administering anappropriate recombinant FVIII product to the subject based on theresults. The hemostatic disorder can be congenital hemophilia A. Thehemostatic disorder can be acquired hemophilia A.

Disclosed is a method for rapidly diagnosing a FVIII haplotype in asubject, comprising, obtaining a sample from the subject, analyzing thesample using rapid PCR, and determining a FVIII haplotype for thesubject. The FVIII haplotype can be selected from the group consistingof H1 (SEQ ID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ IDNO: 4), H5 (SEQ ID NO: 5), or H6 (SEQ ID NO: 6). The subject can bediagnosed with congenital hemophilia A. The subject can be diagnosedwith acquired hemophilia A.

Disclosed is a method of maximizing the sensitivity and specificity ofclinical diagnostic algorithms for identifying a subject with aprothrombotic hemostatic disorder comprising, obtaining a sample fromthe subject, determining whether the FVIII haplotype of a subject havinga hemostatic disorder is H1 (SEQ ID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQID NO: 3), H4 (SEQ ID NO: 4), H5 (SEQ ID NO: 5), or H6 (SEQ ID NO: 6)and performing the appropriate additional laboratory diagnostic testingon the subject based on the results.

Disclosed is a method of treating a subject with a prothrombotichemostatic disorder comprising, identifying a subject with a hemostaticdisorder, determining the FVIII haplotype of the subject, wherein thehaplotype is H1 (SEQ ID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4(SEQ ID NO: 4), H5 (SEQ ID NO: 5), or H6 (SEQ ID NO: 6) andadministering an appropriate anti-thrombotic prophylactic treatmentregimen to the subject based on the results.

Disclosed are antibodies that target high risk haplotypes of FVIII.Disclosed are antibodies that target peptide regions unique to said highrisk haplotypes. Disclosed are agents that neutralize the activity ofhigh risk haplotypes. An agent can be, for example, a monoclonalantibody. Disclosed is an antibody to a polypeptide comprising thesequence as set forth in SEQ ID NO: 19. Disclosed is an n antibody to apolypeptide comprising the sequence as set forth in SEQ ID NO: 20.Disclosed is an n antibody to a polypeptide comprising the sequence asset forth in SEQ ID NO: 21. Disclosed is an n antibody to a polypeptidecomprising the sequence as set forth in SEQ ID NO: 22. Disclosed is anantibody to a polypeptide comprising the sequence as set forth in SEQ IDNO: 23. Disclosed is an antibody to a polypeptide comprising thesequence as set forth in SEQ ID NO: 24.

Disclosed is a method of treating a subject with a prothrombotichemostatic disorder comprising, identifying a subject with aprothrombotic hemostatic disorder, determining the FVIII haplotype ofthe subject, wherein the haplotype is H1 (SEQ ID NO: 1), H2 (SEQ ID NO:2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO: 4), H5 (SEQ ID NO: 5), or H6 (SEQID NO: 6) and administering an appropriate anti-thrombotic prophylactictreatment regimen to the subject based on the results. The hemostaticdisorder can be congenital hemophilia A. The hemostatic disorder can beacquired hemophilia A.

Disclosed is a method of treating a subject with a hemostatic disordercomprising, identifying a subject with a hemostatic disorder,determining the FVIII haplotype of the subject, wherein the haplotype isH1 (SEQ ID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO:4), H5 (SEQ ID NO: 5), or H6 (SEQ ID NO: 6), and administering anappropriate recombinant FVIII product to the subject based on theresults.

Disclosed is a method for rapidly diagnosing a FVIII haplotype in asubject, comprising, obtaining a sample from the subject, analyzing thesample using FVIII haplotype specific antibodies, and determining aFVIII haplotype for the subject. The haplotype can be selected from thegroup consisting of H1 (SEQ ID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO:3), H4 (SEQ ID NO: 4), H5 (SEQ ID NO: 5), or H6 (SEQ ID NO: 6). Thesubject can be diagnosed as having an increased risk for a thromboticevent.

Disclosed is a method of screening to determine the presence ofalloantibodies of FVIII comprising, administering FVIII of a knownhaplotype wherein the haplotype is H1

(SEQ ID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO: 4),H5 (SEQ ID NO: 5), H6 (SEQ ID NO: 6), and determining whetheralloantibody binding occurs.

Disclosed is a method of in vitro screening to determine the presence ofalloantibodies or autoantibodies of FVIII comprising, testing FVIII of aknown haplotype wherein the haplotype is H1 (SEQ ID NO: 1), H2 (SEQ IDNO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO: 4), H5 (SEQ ID NO: 5), H6 (SEQID NO: 6), in a Bethesda Assay for determining the Bethesda Units as ameasure of reactivity with alloantibodies.

Disclosed is an oligonucleotide comprising the sequence as set forth inSEQ ID NO: 1. Disclosed is an oligonucleotide comprising the sequence asset forth in SEQ ID NO: 2. Disclosed is an oligonucleotide comprisingthe sequence as set forth in SEQ ID NO: 3. Disclosed is anoligonucleotide comprising the sequence as set forth in SEQ ID NO: 4.Disclosed is an oligonucleotide comprising the sequence as set forth inSEQ ID NO: 5. Disclosed is an oligonucleotide comprising the sequence asset forth in SEQ ID NO: 6.

Disclosed is an oligonucleotide comprising a sequence selected from thegroup consisting of SEQ ID NO:1-12 and 25-124.

Disclosed is a mixture of primers comprising SEQ ID NOS: 25 and 26.Disclosed is a mixture of primers comprising SEQ ID NOS: 29 and 30.Disclosed is a mixture of primers comprising SEQ ID NOS: 33 and 34.Disclosed is a mixture of primers comprising SEQ ID NOS: 37 and 38.Disclosed is a mixture of primers comprising SEQ ID NOS: 25 and 26.Disclosed is a mixture of primers comprising SEQ ID NOS: 41 and 42.Disclosed is a mixture of primers comprising SEQ ID NOS: 43 and 44.Disclosed is a mixture of primers comprising SEQ ID NOS: 45 and 46.Disclosed is a mixture of primers comprising SEQ ID NOS: 47 and 48.Disclosed is a mixture of primers comprising SEQ ID NOS: 49-124.

Disclosed is a polypeptide comprising the sequence as set forth in SEQID NO: 13. Disclosed is a polypeptide comprising the sequence as setforth in SEQ ID NO: 14. Disclosed is a polypeptide comprising thesequence as set forth in SEQ ID NO: 15. Disclosed is a polypeptidecomprising the sequence as set forth in SEQ ID NO: 16. Disclosed is apolypeptide comprising the sequence as set forth in SEQ ID NO: 17.Disclosed is a polypeptide comprising the sequence as set forth in SEQID NO: 18. Disclosed is a polypeptide comprising the sequence as setforth in SEQ ID NO: 19. Disclosed is a polypeptide comprising thesequence as set forth in SEQ ID NO: 20. Disclosed is a polypeptidecomprising the sequence as set forth in SEQ ID NO: 21. Disclosed is apolypeptide comprising the sequence as set forth in SEQ ID NO: 22.Disclosed is a polypeptide comprising the sequence as set forth in SEQID NO: 23. Disclosed is a polypeptide comprising the sequence as setforth in SEQ ID NO: 24.

Disclosed is a composition comprising pooled FVIII having a singlehaplotype. The haplotype can be H1, H2, H3, H4, H5 or H6.

Disclosed is a recombinant FVIII having the H1 haplotype. Disclosed is arecombinant FVIII having the H2 haplotype. Disclosed is a recombinantFVIII having the H3 haplotype. Disclosed is a recombinant FVIII havingthe H4 haplotype. Disclosed is a recombinant FVIII having the H5haplotype. Disclosed is a recombinant FVIII having the H6 haplotype.

III. BRIEF DESCRIPTION OF THE DRAWINGS

The accompanying drawings, which are incorporated in and constitute apart of this specification, illustrate several embodiments and togetherwith the description illustrate the disclosed compositions and methods.

FIG. 1 is an amplicon model for resequencing an FVIII gene to identifyhaplotype.

FIG. 2 is a schematic representation of non-synonymous single nucleotidepolymorphisms (SNP's) of the human coagulation FVIII protein.

FIG. 3 is a schematic representation of seven wildtype FVIII proteinsand the percentage of racial populations that express such haplotypes.

FIG. 4 is a schematic representation of three commercially availableforms of wildtype FVIII.

FIG. 5 illustrates plasmids that can express five forms of FVIII.

FIG. 6 is a schematic representation of common non-hemophilic F8 nsSNPsindicative of thrombosis susceptibility.

FIG. 7 presents the FVIII:C levels versus age indicating the sex of eachsubject.

FIGS. 8 A, B, and C present the estimated r² and D′ between each pair ofalleles, respectively.

FIG. 9 illustrates the pattern and degree of LD across F8 in varioushuman populations.

IV. DETAILED DESCRIPTION

Before the present compounds, compositions, articles, devices, and/ormethods are disclosed and described, it is to be understood that theyare not limited to specific synthetic methods or specific recombinantbiotechnology methods unless otherwise specified, or to particularreagents unless otherwise specified, as such may, of course, vary. It isalso to be understood that the terminology used herein is for thepurpose of describing particular embodiments only and is not intended tobe limiting.

A. Definitions

As used in the specification and the appended claims, the singular forms“a,” “an” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a pharmaceuticalcarrier” includes mixtures of two or more such carriers, and the like.

Ranges can be expressed herein as from “about” one particular value,and/or to “about” another particular value. When such a range isexpressed, another embodiment includes from the one particular valueand/or to the other particular value. Similarly, when values areexpressed as approximations, by use of the antecedent “about,” it willbe understood that the particular value forms another embodiment. Itwill be further understood that the endpoints of each of the ranges aresignificant both in relation to the other endpoint, and independently ofthe other endpoint. It is also understood that there are a number ofvalues disclosed herein, and that each value is also herein disclosed as“about” that particular value in addition to the value itself. Forexample, if the value “10” is disclosed, then “about 10” is alsodisclosed. It is also understood that when a value is disclosed that“less than or equal to” the value, “greater than or equal to the value”and possible ranges between values are also disclosed, as appropriatelyunderstood by the skilled artisan. For example, if the value “10” isdisclosed the “less than or equal to 10” as well as “greater than orequal to 10” is also disclosed. It is also understood that thethroughout the application, data is provided in a number of differentformats, and that this data, represents endpoints and starting points,and ranges for any combination of the data points. For example, if aparticular data point “10” and a particular data point 15 are disclosed,it is understood that greater than, greater than or equal to, less than,less than or equal to, and equal to 10 and 15 are considered disclosedas well as between 10 and 15. It is also understood that each unitbetween two particular units are also disclosed. For example, if 10 and15 are disclosed, then 11, 12, 13, and 14 are also disclosed.

In this specification and in the claims which follow, reference will bemade to a number of terms which shall be defined to have the followingmeanings:

“Optional” or “optionally” means that the subsequently described eventor circumstance may or may not occur, and that the description includesinstances where said event or circumstance occurs and instances where itdoes not.

“Probes” are molecules capable of interacting with a target nucleicacid, typically in a sequence specific manner, for example throughhybridization. The hybridization of nucleic acids is well understood inthe art and discussed herein. Typically a probe can be made from anycombination of nucleotides or nucleotide derivatives or analogsavailable in the art.

“Primers” are a subset of probes which are capable of supporting sometype of enzymatic manipulation and which can hybridize with a targetnucleic acid such that the t5 enzymatic manipulation can occur. A primercan be made from any combination of nucleotides or nucleotidederivatives or analogs available in the art which do not interfere withthe enzymatic manipulation.

The terms “higher,” “increases,” “elevates,” or “elevation” refer toincreases above basal levels, e.g., as compared to a control. The terms“low,” “lower,” “reduces,” or “reduction” refer to decreases below basallevels, e.g., as compared to a control. For example, basal levels arenormal in vivo levels prior to, or in the absence of, treatment asdisclosed herein.

As used throughout, by a “subject” is meant an individual. Thus, the“subject” can include domesticated animals, such as cats, dogs, etc.,livestock (e.g., cattle, horses, pigs, sheep, goats, etc.), laboratoryanimals (e.g., mouse, rabbit, rat, guinea pig, etc.) and birds.Preferably, the subject is a mammal such as a primate, and, morepreferably, a human.

Throughout this application, various publications are referenced. Thedisclosures of these publications in their entireties are herebyincorporated by reference into this application in order to more fullydescribe the state of the art to which this pertains. The referencesdisclosed are also individually and specifically incorporated byreference herein for the material contained in them that is discussed inthe sentence in which the reference is relied upon.

B. General

The present invention may be understood more readily by reference to thefollowing detailed description of preferred embodiments of the inventionand the Examples included therein and to the Figures and their previousand following description.

Blood clotting begins when platelets adhere to the cut wall of aninjured blood vessel at a lesion site. Subsequently, in a cascade ofenzymatically regulated reactions, soluble fibrinogen molecules areconverted by the enzyme thrombin to insoluble strands of fibrin thathold the platelets together in a thrombus. At each step in the cascade,a protein precursor is converted to a protease that cleaves the nextprotein precursor in the series. Co-factors are required at most of thesteps. Factor VIII circulates as an inactive precursor in blood, boundtightly and non-covalently to von Willebrand factor. Factor VIII isproteolytically activated by thrombin or factor Xa, which dissociates itfrom von Willebrand factor and activates its procoagulant function inthe cascade. In its active form, the protein factor VIIIa is a cofactorthat increases the catalytic efficiency of factor IXa toward factor Xactivation by several orders of magnitude.

People with deficiencies in factor VIII or antibodies against factorVIII who are not treated with factor VIII suffer uncontrolled internalbleeding that may cause a range of serious symptoms, from inflammatoryreactions in joints to early death. Severe hemophiliacs, who numberabout 10,000 in the United States, can be treated with infusion of humanfactor VIII, which will restore the blood's normal clotting ability ifadministered with sufficient frequency and concentration. The classicaldefinition of factor VIII is that substance present in normal bloodplasma that corrects the clotting defect in plasma derived fromindividuals with hemophilia A.

The development of factor VDT (FVIII) inhibitors has been, next to HIVand hepatitis, the most serious complication of hemophilia therapy.Although the recent production of highly purified and geneticallyengineered FVIII products has decreased the risk of these infections,the development of inhibitors remains a major therapeutic challenge.Because affected patients, usually children, are rendered resistant toconventional replacement therapy, control of hemostasis becomesdifficult, resulting in substantial morbidity.

Inhibitors (alloantibodies) are IgG antibodies, mostly of the IgG4subclass, that react with FVIII and interfere with its pro-coagulantfunction. Clinically, patients with inhibitors are classified into highand low responders according to the strength of the anamnestic responsethey experience when they are re-exposed to FVIII. The goals of therapyin these patients are to control severe acute bleeding and to eradicatethe inhibitor.

Two so called by passing agents are used to control bleeding inhemophilia A patients refractory to fVIII. FEIBA, an acronym for “factoreight inhibitor bypassing activity” is derived from large pools of humanplasma and is comprised of activated coagulation factors—prothrombin,factors VIII, IX and X. Considerable care and experience is required touse FEIBA safely. It has been associated with thromboembolic events inpatients, notably disseminated intravascular coagulation with mortalityand at least 14 reported cases of myocardial infarcts. NovoSeven is arecombinant form of activated coagulation factor VIII (rfIII). Theproduct is safe and for most but not all hemophilia A patients effectivein controlling bleeds. A drawback of using any bypassing agent,including NovoSeven, is the absence of reliable quantitative assays tomonitor activity level. In contrast, FVIII levels and activity may bereadily determined and treatment adjusted accordingly. Perhaps the majordisadvantage of NovoSeven is its costs. It has a very short half lifewhich necessitates multiple intravenous infusions. Use of NovoSeven tocover surgery may cost hundreds of thousands of dollars per procedure. Athird product designed to treat inhibitor patients, recombinant porcinefVIII, is in phase 2 clinical trials (www.octagen.com) and not yetcommercially available. Attempts have been made to ascertain the costsof treating high titer inhibitor patients, especially in comparison tothose having no, or low titer. In one study of treatment costs for 104hemophilia patients at Centre Hospitalier Regional Iniversitaire, Lille,France, it was determined that over the period 1988-1995 average annualcosts for treatment were $41,000 for patients having no inhibitors,$46,000 for those having low responding inhibitors and $59,000 for thosewith high responding inhibitors (Goudemand, J 1998).

Another strategy for coping with inhibitors is to attempt to induceimmune tolerance (ITI) to FVIII. ITI involves frequent exposure to FVIIIover extended periods of time and is not always successful. In oneprotocol high purity factor was given at a dose of 100 IU FVIII/kg bodyweight per day for 8-18 months Rocino et, al 1999). The Bonn protocolrequires high doses of FVIII twice daily (Kreuz et. al., 2003). Thelarge amounts of factor needed for successful ITI render it costprohibitive in many circumstances

The pathogenesis of inhibitor development is complex and poorlyunderstood. The nature of the hemophilic mutation is one risk factor forinhibitor development. Patients with large gene deletions, nonsensemutations and intrachromosomal aberrations have a higher incidence ofinhibitors than those with missense mutations, smalldeletions/insertions and splice site mutations. This risk variability isthought to reflect the degree of tolerance that the patient has for theinfused replacement product. A weak correlation has also been shownbetween risk of inhibitor development and major histocompatibilitycomplex (MHC) class I/II genotypes. However, none of these relationshipsis inviolate in that some patients with high risk mutations andunfavorable genotypes do not develop inhibitors, while others withmissense mutations do.

Ethnicity is also a well established risk determinant for inhibitors.The incidence of inhibitors among African-American (AA) hemophiliapatients is twice that observed in Caucasians. No clear explanation ofthe basis for this elevated risk has emerged. Recently, we sequenced theF8 from 137 healthy people representing seven ethnic groups andidentified four common nonsynonymous single nucleotide polymorphisms(nsSNPs). We further found that naturally-occurring haplotypes of thesensSNPs encode six structurally-distinct wildtype FVIII proteins. Five ofthese haplotypes, designated H1, H2, H3, H4 & H5, are expressed byAfrican-Americans, whereas only two, H1 & H2, are expressed byCaucasians. Two haplotypes, H3 & 115, which together are expressed in˜23% of African-Americans, have the minor allele of M2238V in the C2dominant epitope.

Loss-of-function mutations in the gene encoding factor FVIB representthe inherited basis for hemophilia (H)A. Currently, HA is treatedprimarily through exogenous factor replacement therapy using eitherrecombinant (r)—or plasma-derived (pd)-FVIII. There are currently threecommercially available concentrated r-FVIII preparations that are incurrent use for treating HA patients (FIG. 4). As shown in FIG. 4, theH1 and H2 haplotypes are represented by commercially available FVIII.While FVIII has previously been thought to be a monomorphic protein inthe non-hemophilic population, the present invention provides at leastfour common non-synonymous-single-nucleotide-polymorphisms (nsSNPs),combinations of which represent six naturally-occurring allelic variantsof the FVIII protein in the human population (FIG. 2). In FIG. 2, thefive SNP's (W255C, R484H, R776G, D1241E, and M2238V) are illustrated.Combinations of these four SNP's correspond to six haplotypes. This hasbeen determined by direct DNA sequencing of PCR amplified fragments ofthe FVIII genes from numerous unrelated individuals of multipleethnicities; FIG. 1 shows a description of an nsSNP genotyping assaywhich is based on DNA sequencing. PCR can generate 35 amplicons whichare subsequently subjected to automated DNA sequencing. By examiningmale members of different ethnic groups (e.g. because they only have oneX-Chromosome) and females who are homozygous for all nsSNPs or are onlysingly heterozygous, the naturally-occurring haplotypes (H) of thesevariations have been defined (e.g. the combinations by which the allelesof these five nsSNPs segregate naturally). As such, six differenthaplotypic forms of the wt FVIII protein have been identified. Thesehaplotypic forms have been designated: H1 (SEQ ID NO: 1), H2 (SEQ ID NO:2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO: 4), H5 (SEQ ID NO: 5), and H6 (SEQID NO: 6) (FIG. 3). Each of these variants represents a normal allelicvariant of the FVIII protein since the individuals from whom thesequences were described have no bleeding disorders.

Based upon these allelic or haplotypic variants, a specific genetic testhas been Z5 designed to establish the fVIII genotype of any individual(FIG. 1 shows a description of a SNP assay). In addition, theplasmid-based FVIII expression vectors for expressing the differenthaplotypes (e.g. each of these variants) have been developed usingrecombinant technology (FIG. 5). Thus, novel alleles can be identified,cloned into expression vectors, and a screening test developed todetermine the genotype of a given individual. This allows for thedetermination of any subject's allelic type and correct allelic matchingof the replacement FVIII product.

One of the main problems that arise during FVIII replacement therapy isthat the hemophilic recipient often mounts an alloimmune responseagainst the recombinant FVIII protein that is infused. This occursbecause wild type FVIII represents a foreign protein and is thus notrecognized as self by the immune system. In the past, it has beenassumed that this immune response is directed against the wild typesequence(s) that is or are absent in the hemophilic patient due to thepathogenic mutation. However, any subject who has a hemophilic mutationon the background of a different naturally occurring allele than therecombinant factor can also mount an immune response against thenaturally occurring variation(s).

An advantage of the present invention is that matching the replacementproduct to the background allele of the recipient can minimize theimmunological barriers involved in FVIII replacement. In this way, theonly difference is the pathogenic mutation itself. Consequently, theefficacy of FVIII replacement therapy can be maximized and thelikelihood of developing potentially fatal antibody-based inhibitors canbe significantly reduced. This is a clear advantage to the currenttechnology, which is limited by the fact that only two allelic variantof recombinant FVIII is available. Similarly, plasma derived productsare expected to be highly enriched with haplotypes most common in theplasma or blood donor population from which the replacement factor isprocessed. In the US plasma derived preparations of fVIII would beexpected to be somewhat heterogenous with respect to fVIII haplotypecontent but highly enriched for H1. Thus, the present invention allowsexpression of different recombinant FVIII alleles or the use of theexisting recombinant or plasma derived allelic variants to treat matchedsubjects. By matching these alleles to the background allele of thehemophilic subject, the problem of the generation of antibodies thatinhibit successful treatment of hemophilia with recombinant FVIII may bereduced or eliminated.

The relevancy of this approach is not limited to replacement transfusiontherapy of hemophilia A patients with congenital deficiencies of FVIII.There are other known hereditary bleeding disorders resulting fromcongenital deficiencies or functional defects in von Willebrand's factorand coagulation factors IX (FIX) and XI (FXI). Since antibody inhibitorsagainst these proteins are known complications of factor replacementtherapy, the disclosed invention is generally applicable to maximizingtransfusion efficacy in the treatment of these other hemostaticdisorders.

In addition to the problems inherent to congenital hemostatic disorders,this general approach has broad relevancy to treatment of acquiredbleeding diathesis. Transfusion of fresh frozen plasma (FFP) is oftenused in the treatment of bleeding patients with multiple coagulationderangements, such as individuals in liver failure who have decreasedsynthesis of all coagulation factors except FVIII. Since there arelikely to be naturally occurring allelic variants of these othercoagulation factors, analogous to the alleles off FVIII and FIX, suchsubjects can respond by producing inhibitory antibodies reactive againsteach allelic variant not encoded by their own genome. Thus, in themultiply transfused subject, the efficacy of plasma products cansignificantly decline as immunity arises. In contrast to the situationfor cellular transfusion therapy, there is no current methodology formatching plasma donor products to the genotype or haplotype of arecipient. The assay systems disclosed herein, however, allow genotypingof plasma donors and recipients. By matching plasma products to thesubjects receiving them, as is already done with blood cells, theproduction of antibody-based coagulation inhibitors is avoided. This candrastically improve efficacy in the treatment of acquired bleedingdisorders.

Since gene replacement is another approach for factor replacement inhemophilia A and other heritable bleeding diatheses, matching theexpressed coagulation factor with the recipient allele is of utmostimportance at the DNA level for designing various recombinant expressionvectors. The series of different FVIII constructs disclosed, whichrepresent all naturally occurring haplotypic alleles, allow eachhemophiliac undergoing gene therapy to receive allelically matchedreplacement FVIII protein. Therefore, this approach can reduce orprevent the induction of immune responses to the protein whether theexposure occurs by replacement therapy or gone therapy. This isimportant because such a response in the gene therapy setting can resultin both neutralizing antibodies against the protein and lytic responsesagainst host tissues that are successfully transduced with the genetherapy vector. Preventing such a response is an essential requirementfor the success of any gene therapy approach regardless of whichmolecular or cellular vehicle is ultimately found to be the optimalvector for transgene delivery.

Autoantibodies occurring in subjects with acquired hemophilia A differin many aspects from alloantibodies developing in subjects withcongenital hemophilia A after replacement therapy. Like thealloantibodies occurring in severe hemophilia A, factor VIII inhibitorshave been characterized as being predominantly polyclonal, belonging toan IgG4 subclass. However, in contrast to the situation in congenitalhemophilia, monoclonal IgA or IgM antibodies have also been described insubjects with acquired hemophilia A associated with hematologicmalignancies. Another difference between FVIII autoantibodies andalloantibodies lies in their method of inhibition. The differences are,however, subtle, as autoantibody inhibitors are mainly directed againstsingle epitopes on the factor VIII molecule (A2 domain, A3 domain, and,more frequently, C2 domain), whereas alloantibodies are usually directedagainst both the A2 and C2 domains and sometimes against the A3 domain.

Epitopes that are immunoreactive with antibodies that inhibit thecoagulant activity of factor VIII have been characterized based on knownstructure-function relationships in factor VIII. Inhibitors can, forexample, act by disrupting any of the macromolecular interactionsassociated with the domain structure of factor VIII or its associationswith von Willebrand factor, thrombin, factor Xa, factor IXa, or factorX. However, most inhibitory antibodies to human factor VIII act bybinding to epitopes located in the 40 kDa A2 domain or 20 kDa C2 domainof factor VIII, disrupting specific functions associated with thesedomains, as described by Fulcher et al. (1985) Proc. Natl. Acad. Sci.USA 82:7728-7732; and Scandella et al. (1988) Proc. Natl. Acad. Sci. USA85:6152-6156 (herein incorporated by reference in their entirety). Inaddition to the A2 and C2 epitopes, there may be a third epitope in theA3 or C1 domain of the light chain of factor VIII, according toScandella et al. (1993) Blood 82:1767-1775. The significance of thisputative third epitope is unknown, but it appears to account for a minorfraction of the epitope reactivity in factor VIII.

Anti-A2 antibodies block factor X activation, as shown by Lollar et al.(1994) J. Clin. Invest. 93:2497-2504 (incorporated by reference in theirentirety). Previous mapping studies by deletion mutagenesis described byWare et al. (1992) Blood Coagul. Fibrinolysis 3:703-716, incorporated byreference in their entirety, located the A2 epitope to within a 20 kDaregion of the NH.sub.2-terminal end of the 40 kDa A2 domain. Competitionimmunoradiometric assays have indicated that A2 inhibitors recognizeeither a common epitope or narrowly clustered epitopes, as described byScandella et al. (1992) Throm. Haemostas. 67:665-671, and asdemonstrated in U.S. Pat. No. 5,859,204 (both herein incorporated byreference in their entirety).

In this application the term low, reduced, or nonantigenic FVIII is usedto describe a FVIII construct that does not demonstrate reducedcoagulant activity in vitro when exposed to potentially antibodies or invivo when administrated to a patient that has deficient FVIII coagulantactivity. The patient may have congenital or acquired hemophilia A.

In this application the term low, reduced or nonimmunogenic FVIII isused to describe a FVIII construct that when administered to previouslyuntreated congenital hemophilia A patients does not stimulate theformation of antibodies that react with such fVIII construct orstimulates the formation of antibodies that react with such factor in aless effective manner or in a manner that does not reduce the clinicalefficacy of the administered factor to the same degree as otherreplacement fVIII do.

Testing of Recombinant Factor VIII Molecules: Factor VIII replacementmolecules that are haplotypically matched to the patient haplotype(haplotypically matched fVIII) can be tested in humans for their reducedantigenicity and/or immunogenicity in at least two types of clinicaltrials. In one type of trial, designed to determine whetherhaplotypically matched factor VIII is immunoreactive with inhibitoryantibodies haplotypically matched factor VIII s administered, preferablyby intravenous infusion, to approximately 25 patients having factor VIIIdeficiency who have antibodies to factor VIII that inhibit the coagulantactivity of therapeutic human FVII that is not haplotypically matched.The dosage of haplotypically matched fVIII is in a range between 5 and50 Units/kg body weight, preferably 10-50 Units/kg, and most preferably40 Units/kg body weight. Approximately 1 hour after each administration,the recovery of factor VIII from blood samples is measured in aone-stage coagulation assay. Samples are taken again approximately 5hours after infusion, and recovery is measured. Total recovery and therate of disappearance of factor VIII from the samples is predictive ofthe antibody titer and inhibitory activity. If the antibody titer ishigh, factor VIII recovery usually cannot be measured. The recoveryresults of the haplotypically matched fVIII are compared to the recoveryof recovery results for factor VIII, that is not haplotypically matchedporcine factor VIII, and other commonly used therapeutic forms of factorVIII or factor VIII substitutes.

In a second type of clinical trial, designed to determine whether thehaplotypically matched fVIII is immunogenic, i.e., whether patients willdevelop inhibitory antibodies that react with the haplotypically matchedfVIII, haplotypically matched fVIII, is administered, as described inthe preceding paragraph, to approximately 100 previously untreatedhemophiliac patients who have not developed antibodies to factor VIII.Treatments are given approximately every 2 weeks over a period of 6months to 1 year. At 1 to 3 month intervals during this period, bloodsamples are drawn and Bethesda assays or other antibody assays areperformed to determine the presence of inhibitory antibodies. Recoveryassays can also be done, as described above, after each infusion.Results are compared to hemophiliac patients who receive human factorVIII that is not haplotypically matched or to porcine factor VIII, orother commonly used therapeutic forms of factor VIII or factor VIIIsubstitutes.

Anti-FVIII antibodies (Ab) include three subtypes analogous to RBCantigens (Ag) and Ab. The three subtypes include:

-   -   Isoantibodies→the subtype of anti-FVIII Ab that occurs in        hemophilia A patients who have no immunologically detectable        circulating FVIII protein. This type of MB-deficiency is        referred to as cross-reactive-material-negative (CRM-), and is        due to the presence of a F8 mutation subtype known as a        null-mutation, which include the following: large deletions,        nonsense mutations and intragenic inversions, such as the        recurrent intron 22 inversion, or, less frequently, the intron 1        inversion.    -   Alloantibodies→the subtype of anti-FVIII Ab that occurs in        hemophilia A patients who have an immunologically detectable        circulating FVIII protein that is dysfunctional. This type of        FVIII-deficiency is referred to as either CRM-positive (CRM+) or        CRM-reduced (CRM-r), and is typically found in patients who have        a hemophilic missense F8 mutation. Hemophilia A patients with        certain small deletions and/or splice-site mutations can also        have CRM+ or CRM-r FVIII-deficiencies.    -   Autoantibodies→the subtype of anti-FVIII Ab that occurs in        non-hemophilic individuals with wildtype FVIII who develop        acquired hemophilia A.        These anti-FVIII antibodies bind and inhibit the function of        FVIII (i.e. the catalytically inactive form of the molecule        referred to as the pro-cofactor form) and/or FVIIIa (i.e. the        catalytically active form of the molecule referred to as the        cofactor form), both in vivo and in clotting assays in vitro.        The antibodies are usually IgG (primarily IgG4) and bind limited        regions of FVIII. A subset of the anti-FVIII Ab that develop can        increase peripheral clearance and/or degradation of FVIII.

While several effective pharmacologic agents are already available toclinicians for use in both treating and prophylaxing against thrombosis,physicians are not able to accurately identify those individuals atgreatest risk for these disorders. Since strokes and heart attacks arejust two facets of thrombosis, many more people will suffer from thisdisorder than are affected by hemophilia A, or from all congenitalbleeding disorders combined. Disclosed are diagnostic algorithms, whichaccurately identify at risk individuals and allow prophylactic riskreduction regimens to be implemented prior to their manifesting a strokeor heart attack, or other types cardiovascular thrombosis. The allelesof the four ns-SNPs, which underlie the 6 naturally-occurring forms ofthe FVIII protein in humans, are functionally distinct and may influencethe circulating level of FVIII. With respect to the D1241E ns-SNP, forexample, those with the E-allele at the protein level (or G-allele atthe nucleotide level), have about a 25% lower mean circulating FVIIIlevel. Since elevated circulating levels of this coagulation protein afrequently observed phenotype in the non-hemophilic population, havebeen associated with elevated risk for both arterial and venousthrombosis, the E-allele of D1241E may be protective against thrombosis.Therefore, the methods disclosed for differentiating between the allelesof, for example, the D1241E ns-SNP, or, within the set of sixnaturally-occurring haplotypes of the FVIII protein, the subset thatcontains the E-allele of this ns-SNP (i.e. H2, H3, H4, and H6) and thesubset that contains the D-allele (i.e. H1 and H5) improve currentdiagnostic algorithms for assessing thrombosis risk. This type oftesting, in contrast to the rapid testing described herein for thehemophilia, is largely for diagnosic risk assessment, in order to guideprophylactic treatment decisions, as such does not necessarily have tobe performed in a rapid manner.

The present invention is described with regards to Hemophilia as well asother hemostatic disorders including: afibrinogenemia,dysfibrinogenemia, nonplatelet hemostasis, coagulation, thrombosis,thrombophilia, FV deficiency, Owren disease, parahemophilia, FVIIdeficiency, FVIII deficiency, FXI deficiency, FXI deficiency, FXIIdeficiency, FXIII deficiency, factor V Leiden deficiency, DIC, protein Cdeficiency, activated protein C resistance, protein S deficiency,antithrombin III deficiency, hypoprothrombinemias, cryoglobulinemias,multiple myeloma, Waldenstrom macroglobulinemia, Henoch-Schönleinpurpura, hyperglobulinemic purpura, cavernous hemangioma, hereditaryhemorrhagic telangiectasia, pseudoxanthoma elasticum, Ehlers-Danlossyndrome, Cushing syndrome, Shwartzman phenomenon, von Willebranddisease, Waterhouse-Friderichsen syndrome, and Wiskott-Aldrich syndrome.

Thus, disclosed herein are methods and compositions relating to thevarious haplotypes of FVIII, and how they can be used advantageously totreat subjects with diseases and disorders relating to FVIII.

C. Compositions

Disclosed are the components to be used to prepare the disclosedcompositions as well as the compositions themselves to be used withinthe methods disclosed herein. For example, disclosed herein are varioushaplotypes of FVIII, each with a different sequence identity. Thesehaplotypes and variations and combinations of these haplotypes arecontemplated. It should be noted that the specific haplotypes disclosedherein need not be varied, and their specific sequences are importantwith many of the methods disclosed herein. However, where variations canoccur, and where such variations are useful, the following principlesregarding homology and sequence variation apply. Therefore, it isunderstood that when combinations, subsets, interactions, groups, etc.of these materials are disclosed that while specific reference of eachvarious individual and collective combinations and permutation of thesecompounds may not be explicitly disclosed, each is specificallycontemplated and described herein, wherein such may apply. For example,if a particular haplotype is disclosed and discussed and a number ofmodifications that can be made to a number of molecules including thepoint of interest of the haplotype are discussed, specificallycontemplated is each and every combination and permutation of thehaplotype and the modifications that are possible unless specificallyindicated to the contrary. Thus, if a class of molecules A, B, and C aredisclosed as well as a class of molecules D, E, and F and an example ofa combination molecule, A-D is disclosed, then even if each is notindividually recited each is individually and collectively contemplatedmeaning combinations, A-E, A-F, B-D, B-E, B-F, C-D, C-E, and C-F areconsidered disclosed. Likewise, any subset or combination of these isalso disclosed. Thus, for example, the sub-group of A-E, B-F, and C-Ewould be considered disclosed. This concept applies to all aspects ofthis application including, but not limited to, steps in methods ofmaking and using the disclosed compositions. Thus, if there are avariety of additional steps that can be performed it is understood thateach of these additional steps can be performed with any specificembodiment or combination of embodiments of the disclosed methods.

1. Sequence Similarities

It is understood that as discussed herein the use of the terms homologyand identity mean the same thing as similarity. Thus, for example, ifthe use of the word homology is used between two non-natural sequencesit is understood that this is not necessarily indicating an evolutionaryrelationship between these two sequences, but rather is looking at thesimilarity or relatedness between their nucleic acid sequences. Many ofthe methods for determining homology between two evolutionarily relatedmolecules are routinely applied to any two or more nucleic acids orproteins for the purpose of measuring sequence similarity regardless ofwhether they are evolutionarily related or not.

In general, it is understood that one way to define any known variantsand derivatives or those that might arise, of the disclosed genes andproteins herein, is through defining the variants and derivatives interms of homology to specific known sequences. This identity ofparticular sequences disclosed herein is also discussed elsewhereherein. In general, variants of genes and proteins herein disclosedtypically can have at least, about 70, 71, 72, 73, 74, 75, 76, 77, 78,79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,97, 98, or 99 percent homology to the stated sequence or the nativesequence. This principle can apply to primers, probes, and other nucleicacids and proteins as described herein. However, in certain instances asdisclosed herein, genetic variants have defined sequence differenceswhich may not have a given percentage of change, but may instead have 1,2, 3, 4, 5, 6, 7, 8, 9, 10, or more differences in nucleic acid or aminoacid sequence when compared to a native, known, or control sequence.Those of skill in the art readily understand how to determine thehomology of two proteins or nucleic acids, such as genes. For example,the homology can be calculated after aligning the two sequences so thatthe homology is at its highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment. It isunderstood that any of the methods typically can be used and that incertain instances the results of these various methods may differ, butthe skilled artisan understands if identity is found with at least oneof these methods, the sequences would be said to have the statedidentity, and be disclosed herein.

For example, as used herein, a sequence recited as having a particularpercent homology to another sequence refers to sequences that have therecited homology as calculated by any one or more of the calculationmethods described above. For example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingthe Zuker calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by any of theother calculation methods. As another example, a first sequence has 80percent homology, as defined herein, to a second sequence if the firstsequence is calculated to have 80 percent homology to the secondsequence using both the Zuker calculation method and the Pearson andLipman calculation method even if the first sequence does not have 80percent homology to the second sequence as calculated by the Smith andWaterman calculation method, the Needleman and Wunsch calculationmethod, the Jaeger calculation methods, or any of the other calculationmethods. As yet another example, a first sequence has 80 percenthomology, as defined herein, to a second sequence if the first sequenceis calculated to have 80 percent homology to the second sequence usingeach of calculation methods (although, in practice, the differentcalculation methods will often result in different calculated homologypercentages).

2. Hybridization/Selective Hybridization

The term hybridization typically means a sequence driven interactionbetween at least two nucleic acid molecules, such as a primer or a probeand a gene. Sequence driven interaction means an interaction that occursbetween two nucleotides or nucleotide analogs or nucleotide derivativesin a nucleotide specific manner. For example, G interacting with C or Ainteracting with T are sequence driven interactions. Typically sequencedriven interactions occur on the Watson-Crick face or Hoogsteen face ofthe nucleotide. The hybridization of two nucleic acids is affected by anumber of conditions and parameters known to those of skill in the art.For example, the salt concentrations, pH, and temperature of thereaction all affect whether two nucleic acid molecules will hybridize.

Parameters for selective hybridization between two nucleic acidmolecules are well known to those of skill in the art. For example, insome embodiments selective hybridization conditions can be defined asstringent hybridization conditions. For example, stringency ofhybridization is controlled by both temperature and salt concentrationof either or both of the hybridization and washing steps. For example,the conditions of hybridization to achieve selective hybridization mayinvolve hybridization in high ionic strength solution (6×SSC or 6×SSPE)at a temperature that is about 12-25° C. below the Tm (the meltingtemperature at which half of the molecules dissociate from theirhybridization partners) followed by washing at a combination oftemperature and salt concentration chosen so that the washingtemperature is about 5° C. to 20° C. below the Tm. The temperature andsalt conditions are readily determined empirically in preliminaryexperiments in which samples of reference DNA immobilized on filters arehybridized to a labeled nucleic acid of interest and then washed underconditions of different stringencies. Hybridization temperatures aretypically higher for DNA-RNA and RNA-RNA hybridizations. The conditionscan be used as described above to achieve stringency, or as is known inthe art. (Sambrook et al., Molecular Cloning: A Laboratory Manual, 2ndEd., Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y., 1989;Kunkel et al. Methods Enzymol. 1987:154:367, 1987 which is hereinincorporated by reference for material at least related to hybridizationof nucleic acids). A preferable stringent hybridization condition for aDNA:DNA hybridization can be at about 68° C. (in aqueous solution) in6×SSC or 6×SSPE followed by washing at 68° C. Stringency ofhybridization and washing, if desired, can be reduced accordingly as thedegree of complementarity desired is decreased, and further, dependingupon the G-C or A-T richness of any area wherein variability is searchedfor. Likewise, stringency of hybridization and washing, if desired, canbe increased accordingly as homology desired is increased, and further,depending upon the G-C or A-T richness of any area wherein high homologyis desired, all as known in the art.

Another way to define selective hybridization is by looking at theamount (percentage) of one of the nucleic acids bound to the othernucleic acid. For example, in some embodiments selective hybridizationconditions would be when at least about, 60, 65, 70, 71, 72, 73, 74, 75,76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93,94, 95, 96, 97, 98, 99, 100 percent of the limiting nucleic acid isbound to the non-limiting nucleic acid. Typically, the non-limitingprimer is in for example, 10 or 100 or 1000 fold excess. This type ofassay can be performed at under conditions where both the limiting andnon-limiting primer are for example, 10 fold or 100 fold or 1000 foldbelow their k_(d), or where only one of the nucleic acid molecules is 10fold or 100 fold or 1000 fold or where one or both nucleic acidmolecules are above their k_(d).

Another way to define selective hybridization is by looking at thepercentage of primer that gets enzymatically manipulated underconditions where hybridization is required to promote the desiredenzymatic manipulation. For example, in some embodiments selectivehybridization conditions would be when at least about, 60, 65, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 percent of the primer isenzymatically manipulated under conditions which promote the enzymaticmanipulation, for example if the enzymatic manipulation is DNAextension, then selective hybridization conditions would be when atleast about 60, 65, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82,83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100percent of the primer molecules are extended. Preferred conditions alsoinclude those suggested by the manufacturer or indicated in the art asbeing appropriate for the enzyme performing the manipulation.

Just as with homology, it is understood that there are a variety ofmethods herein disclosed for determining the level of hybridizationbetween two nucleic acid molecules. It is understood that these methodsand conditions may provide different percentages of hybridizationbetween two nucleic acid molecules, but unless otherwise indicatedmeeting the parameters of any of the methods would be sufficient. Forexample if 80% hybridization was required and as long as hybridizationoccurs within the required parameters in any one of these methods it isconsidered disclosed herein.

It is understood that those of skill in the art understand that if acomposition or method meets any one of these criteria for determininghybridization either collectively or singly it is a composition ormethod that is disclosed herein.

3. Nucleic Acids

There are a variety of molecules disclosed herein that are nucleic acidbased, including for example the nucleic acids that encode, for exampleFVIII, as well as various functional nucleic acids. The disclosednucleic acids are made up of for example, nucleotides, nucleotideanalogs, or nucleotide substitutes. Non-limiting examples of these andother molecules are discussed herein. It is understood that, forexample, when a vector is expressed in a cell, the expressed mRNA willtypically be made up of A, C, G, and U. Likewise, it is understood thatif, for example, an antisense molecule is introduced into a cell or cellenvironment through for example exogenous delivery, it is advantageousthat the antisense molecule be made up of nucleotide analogs that reducethe degradation of the antisense molecule in the cellular environment.

a) Nucleotides and Related Molecules

A nucleotide is a molecule that contains a base moiety, a sugar moietyand a phosphate moiety. Nucleotides can be linked together through theirphosphate moieties and sugar moieties creating an internucleosidelinkage. The base moiety of a nucleotide can be adenin-9-yl (A),cytosin-1-yl (C), guanin-9-yl (G), uracil-1-yl (U), and thymin-1-yl (T).The sugar moiety of a nucleotide is a ribose or a deoxyribose. Thephosphate moiety of a nucleotide is pentavalent phosphate. Annon-limiting example of a nucleotide would be 3′-AMP (3′-adenosinemonophosphate) or 5′-GMP (5′-guanosine monophosphate).

A nucleotide analog is a nucleotide which contains some type ofmodification to either the base, sugar, or phosphate moieties.Modifications to the base moiety would include natural and syntheticmodifications of A, C, G, and T/U as well as different purine orpyrimidine bases, such as uracil-5-yl (.psi.), hypoxanthin-9-yl (I), and2-aminoadenin-9-yl. A modified base includes but is not limited to5-methylcytosine (5-me-C), 5-hydroxymethyl cytosine, xanthine,hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives ofadenine and guanine, 2-propyl and other alkyl derivatives of adenine andguanine, 2-thiouracil, 2-thiothymine and

2-thiocytosine, 5-halouracil and cytosine, 5-propynyl uracil andcytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudouracil),4-thiouracil, 8-halo, 8-amino, 8-thiol, 8-thioalkyl, 8-hydroxyl andother 8-substituted adenines and guanines, 5-halo particularly 5-bromo,5-trifluoromethyl and other 5-substituted uracils and cytosines,7-methylguanine and 7-methyladenine, 8-azaguanine and 8-azaadenine,7-deazaguanine and 7-deazaadenine and 3-deazaguanine and 3-deazaadenine.Additional base modifications can be found for example in U.S. Pat. No.3,687,808, Englisch et al., Angewandte Chemie, International Edition,1991, 30, 613, and Sanghvi, Y. S., Chapter 15, Antisense Research andApplications, pages 289-302, Crooke, S. T. and Lebleu, B. ed., CRCPress, 1993. Certain nucleotide analogs, such as 5-substitutedpyrimidines, 6-azapyrimidines and N-2, N-6 and O-6 substituted purines,including 2-aminopropyladenine, 5-propynyluracil and 5-propynylcytosine.5-methylcytosine can increase the stability of duplex formation. Oftentime base modifications can be combined with for example a sugarmodification, such as 2′-O-methoxyethyl, to achieve unique propertiessuch as increased duplex stability. There are numerous United Statespatents such as U.S. Pat. Nos. 4,845,205; 5,130,302; 5,134,066;5,175,273; 5,367,066; 5,432,272; 5,457,187; 5,459,255; 5,484,908;5,502,177; 5,525,711; 5,552,540; 5,587,469; 5,594,121, 5,596,091;5,614,617; and 5,681,941, which detail and describe a range of basemodifications. Each of these patents is herein incorporated byreference.

Nucleotide analogs can also include modifications of the sugar moiety.Modifications to the sugar moiety would include natural modifications ofthe ribose and deoxy ribose as well as synthetic modifications. Sugarmodifications include but are not limited to the following modificationsat the 2′ position: OH; F; O-, S-, or N-alkyl; O-, S-, or N-alkenyl; O-,S- or N-alkynyl; or O-alkyl-O-alkyl, wherein the alkyl, alkenyl andalkynyl may be substituted or unsubstituted C₁ to C₁₀, alkyl or C₂ toC₁₀ alkenyl and alkynyl. 2′ sugar modifications also include but are notlimited to —O[(CH₂)_(n)O]_(m)CH₃, —O(CH₂)_(n)OCH₃, —O(CH₂)_(n)NH₂,—O(CH₂)_(n)CH₃, —O(CH₂)_(n)—ONH₂, and —O(CH₂)_(n)ON[(CH₂)_(n)CH₃)]₂,where n and m are from 1 to about 10.

Other modifications at the 2′ position include but are not limited to:C₁ to C₁₀ lower alkyl, substituted lower alkyl, alkaryl, aralkyl,O-alkaryl or O-aralkyl, SH, SCH₃, OCN, Cl, Br, CN, CF₃, OCF₃, SOCH₃,SO₂CH₃, ONO₂, NO₂, N₃, NH₂, heterocycloalkyl, heterocycloalkaryl,aminoalkylamino, polyalkylamino, substituted silyl, an RNA cleavinggroup, a reporter group, an intercalator, a group for improving thepharmacokinetic properties of an oligonucleotide, or a group forimproving the pharmacodynamic properties of an oligonucleotide, andother substituents having similar properties. Similar modifications mayalso be made at other positions on the sugar, particularly the 3′position of the sugar on the 3′ terminal nucleotide or in 2′-5′ linkedoligonucleotides and the 5′ position of 5′ terminal nucleotide. Modifiedsugars would also include those that contain modifications at thebridging ring oxygen, such as CH₂ and S, Nucleotide sugar analogs mayalso have sugar mimetics such as cyclobutyl moieties in place of thepentofuranosyl sugar. There are numerous United States patents thatteach the preparation of such modified sugar structures such as U.S.Pat. Nos. 4,981,957; 5,118,800; 5,319,080; 5,359,044; 5,393,878;5,446,137; 5,466,786; 5,514,785; 5,519,134; 5,567,811; 5,576,427;5,591,722; 5,597,909; 5,610,300; 5,627,053; 5,639,873; 5,646,265;5,658,873; 5,670,633; and 5,700,920, each of which is hereinincorporated by reference in its entirety.

Nucleotide analogs can also be modified at the phosphate moiety.Modified phosphate moieties include but are not limited to those thatcan be modified so that the linkage between two nucleotides contains aphosphorothioate, chiral phosphorothioate, phosphorodithioate,phosphotriester, aminoalkylphosphotriester, methyl and other alkylphosphonates including 3′-alkylene phosphonate and chiral phosphonates,phosphinates, phosphoramidates including 3′-amino phosphoramidate andaminoalkylphosphoramidates, thionophosphoramidates,thionoalkylphosphonates, thionoalkylphosphotriesters, andboranophosphates. It is understood that these phosphate or modifiedphosphate linkage between two nucleotides can be through a 3′-5′ linkageor a 2′-5′ linkage, and the linkage can contain inverted polarity suchas 3′-5′ to 5′-3′ or 2′-5′ to 5′-2′. Various salts, mixed salts and freeacid forms are also included. Numerous United States patents teach howto make and use nucleotides containing modified phosphates and includebut are not limited to, U.S. Pat. Nos. 3,687,808; 4,469,863; 4,476,301;5,023,243; 5,177,196; 5,188,897; 5,264,423; 5,276,019; 5,278,302;5,286,717; 5,321,131; 5,399,676; 5,405,939; 5,453,496; 5,455,233;5,466,677; 5,476,925; 5,519,126; 5,536,821; 5,541,306; 5,550,111;5,563,253; 5,571,799; 5,587,361; and 5,625,050, each of which is hereinincorporated by reference.

It is understood that nucleotide analogs need only contain a singlemodification, but may also contain multiple modifications within one ofthe moieties or between different moieties.

Nucleotide substitutes are molecules having similar functionalproperties to nucleotides, but which do not contain a phosphate moiety,such as peptide nucleic acid (PNA). Nucleotide substitutes are moleculesthat will recognize nucleic acids in a Watson-Crick or Hoogsteen manner,but which are linked together through a moiety other than a phosphatemoiety. Nucleotide substitutes are able to conform to a double helixtype structure when interacting with the appropriate target nucleicacid.

Nucleotide substitutes are nucleotides or nucleotide analogs that havehad the phosphate moiety and/or sugar moieties replaced. Nucleotidesubstitutes do not contain a standard phosphorus atom. Substitutes forthe phosphate can be for example, short chain alkyl or cycloalkylinternucleoside linkages, mixed heteroatom and alkyl or cycloalkylinternucleoside linkages, or one or more short chain heteroatomic orheterocyclic internucleoside linkages. These include those havingmorpholino linkages (formed in part from the sugar portion of anucleoside); siloxane backbones; sulfide, sulfoxide and sulfonebackbones; formacetyl and thioformacetyl backbones; methylene formacetyland thioformacetyl backbones; alkene containing backbones; sulfamatebackbones; methyleneimino and methylenehydrazino backbones; sulfonateand sulfonamide backbones; amide backbones; and others having mixed N,O, S and CH₂ component parts. Numerous United States patents disclosehow to make and use these types of phosphate replacements and includebut are not limited to U.S. Pat. Nos. 5,034,506; 5,166,315; 5,185,444;5,214,134; 5,216,141; 5,235,033; 5,264,562; 5,264,564; 5,405,938;5,434,257; 5,466,677; 5,470,967; 5,489,677; 5,541,307; 5,561,225;5,596,086; 5,602,240; 5,610,289; 5,602,240; 5,608,046; 5,610,289;5,618,704; 5,623,070; 5,663,312; 5,633,360; 5,677,437; and 5,677,439,each of which is herein incorporated by reference.

It is also understood in a nucleotide substitute that both the sugar andthe phosphate moieties of the nucleotide can be replaced, by for examplean amide type linkage (aminoethylglycine) (PNA). U.S. Pat. Nos.5,539,082; 5,714,331; and 5,719,262 teach how to make and use PNAmolecules, each of which is herein incorporated by reference. (See alsoNielsen et al., Science, 1991, 254, 1497-1500).

It is also possible to link other types of molecules (conjugates) tonucleotides or nucleotide analogs to enhance for example, cellularuptake. Conjugates can be chemically linked to the nucleotide ornucleotide analogs. Such conjugates include but are not limited to lipidmoieties such as a cholesterol moiety (Letsinger et al., Proc. Natl.Acad. Sci. USA, 1989,

86, 6553-6556), cholic acid (Manoharan et al., Bioorg. Med. Chem. Let.,1994, 4, 1053-1060), a thioether, e.g., hexyl-5-tritylthiol (Manoharanet al., Ann. N.Y. Acad. Sci., 1992, 660, 306-309; Manoharan et al.,Bioorg. Med. Chem. Let., 1993, 3, 2765-2770), a thiocholesterol(Oberhauser et al., Nucl. Acids Res., 1992, 20, 533-538), an aliphaticchain, e.g., dodecandiol or undecyl residues (Saison-Behmoaras et al.,EMBO J., 1991, 10, 1111-1118; Kabanov et al., FEBS Lett., 1990, 259,327-330; Svinarchuk et al., Biochimie, 1993, 75, 49-54), a phospholipid,e.g., di-hexadecyl-rac-glycerol or triethylammonium1,2-di-O-hexadecyl-rac-glycero-3-H-phosphonate (Manoharan et al.,Tetrahedron Lett., 1995, 36, 3651-3654; Shea et al., Nucl. Acids Res.,1990, 18, 3777-3783), a polyamine or a polyethylene glycol chain(Manoharan et al., Nucleosides & Nucleotides, 1995, 14, 969-973), oradamantane acetic acid (Manoharan et al., Tetrahedron Lett., 1995, 36,3651-3654), a palmityl moiety (Mishra et al., Biochim. Biophys. Acta,1995, 1264, 229-237), or an octadecylamine orhexylamino-carbonyl-oxycholesterol moiety (Crooke et al., J. Pharmacol.Exp. Ther., 1996, 277, 923-937. Numerous United States patents teach thepreparation of such conjugates and include, but are not limited to U.S.Pat. Nos. 4,828,979; 4,948,882; 5,218,105; 5,525,465; 5,541,313;5,545,730; 5,552,538; 5,578,717, 5,580,731; 5,580,731; 5,591,584;5,109,124; 5,118,802; 5,138,045; 5,414,077; 5,486,603; 5,512,439;5,578,718; 5,608,046; 4,587,044; 4,605,735; 4,667,025; 4,762,779;4,789,737; 4,824,941; 4,835,263; 4,876,335; 4,904,582; 4,958,013;5,082,830; 5,112,963; 5,214,136; 5,082,830; 5,112,963; 5,214,136;5,245,022; 5,254,469; 5,258,506; 5,262,536; 5,272,250; 5,292,873;5,317,098; 5,371,241, 5,391,723; 5,416,203, 5,451,463; 5,510,475;5,512,667; 5,514,785; 5,565,552; 5,567,810; 5,574,142; 5,585,481;5,587,371; 5,595,726; 5,597,696; 5,599,923; 5,599,928 and 5,688,941,each of which is herein incorporated by reference.

A Watson-Crick interaction is at least one interaction with theWatson-Crick face of a nucleotide, nucleotide analog, or nucleotidesubstitute. The Watson-Crick face of a nucleotide, nucleotide analog, ornucleotide substitute includes the C2, N1, and C6 positions of a purinebased nucleotide, nucleotide analog, or nucleotide substitute and theC2, N3, C4 positions of a pyrimidine based nucleotide, nucleotideanalog, or nucleotide substitute.

A Hoogsteen interaction is the interaction that takes place on theHoogsteen face of a nucleotide or nucleotide analog, which is exposed inthe major groove of duplex DNA. The Hoogsteen face includes the N7position and reactive groups (NH₂ or O) at the C6 position of purinenucleotides.

b) Sequences

There are a variety of sequences related to the FVIII gene, thesesequences and others are attached.

c) Primers and Probes

Disclosed are compositions including primers and probes, which arecapable of interacting with the FVIII gene as disclosed herein. Incertain embodiments the primers are used to support DNA amplificationreactions. Typically the primers will be capable of being extended in asequence specific manner. Extension of a primer in a sequence specificmanner includes any methods wherein the sequence and/or composition ofthe nucleic acid molecule to which the primer is hybridized or otherwiseassociated directs or influences the composition or sequence of theproduct produced by the extension of the primer. Extension of the primerin a sequence specific manner therefore includes, but is not limited to,PCR, DNA sequencing, DNA extension, DNA polymerization, RNAtranscription, or reverse transcription. Techniques and conditions thatamplify the primer in a sequence specific manner are preferred. Incertain embodiments the primers are used for the DNA amplificationreactions, such as PCR or direct sequencing. It is understood that incertain embodiments the primers can also be extended using non-enzymatictechniques, where for example, the nucleotides or oligonucleotides usedto extend the primer are modified such that they will chemically reactto extend the primer in a sequence specific manner. Typically thedisclosed primers hybridize with the FVIII gene or region of the FVIIIgene or they hybridize with the complement of the FVIII gene orcomplement of a region of the FVIII gene.

The size of the primers or probes for interaction with the FVIII gene incertain embodiments can be any size that supports the desired enzymaticmanipulation of the primer, such as DNA amplification or the simplehybridization of the probe or primer. A typical FVIII haplotype primeror probe would be at least 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17,18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35,36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53,54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71,72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89,90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100, 125, 150, 175, 200, 225,250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 550, 600, 650,700, 750, 800, 850, 900, 950, 1000, 1250, 1500, 1750, 2000, 2250, 2500,2750, 3000, 3500, or 4000 nucleotides long.

In other embodiments the primer or probe can be less than or equal to 6,7, 8, 9, 10, 11, 12 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43,44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61,62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79,80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97,98, 99, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400,425, 450, 475, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000,1250, 1500, 1750, 2000, 2250, 2500, 2750, 3000, 3500, or 4000nucleotides long.

The primers for the FVIII gene typically will be used to produce anamplified DNA product that contains the FVIII gene. In general,typically the size of the product will be such that the size can beaccurately determined to within 3, or 2 or 1 nucleotides.

d) Nucleic Acid Delivery

The haplotypes disclosed herein can be useful with various methods ofnucleic acid delivery. For example, in a subject with a given haplotypeof FVIII, the corresponding nucleic acid of that haplotype can beadministered to the subject, thereby increasing the amount of the properhaplotype if FVIII in that particular subject, thereby decreasingadverse reactions to the expressed protein. In the methods describedabove which include the administration and uptake of exogenous DNA intothe cells of a subject (i.e., gene transduction or transfection), thedisclosed nucleic acids can be in the form of naked DNA or RNA, or thenucleic acids can be in a vector for delivering the nucleic acids to thecells, whereby the antibody-encoding DNA fragment is under thetranscriptional regulation of a promoter, as would be well understood byone of ordinary skill in the art. The vector can be a commerciallyavailable preparation, such as an adenovirus vector (QuantumBiotechnologies, Inc. (Laval, Quebec, Canada). Delivery of the nucleicacid or vector to cells can be via a variety of mechanisms. As oneexample, delivery can be via a liposome, using commercially availableliposome preparations such as LIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL,Inc., Gaithersburg, Md.), SUPERFECT (Qiagen, Inc. Hilden, Germany) andTRANSFECTAM (Promega Biotec, Inc., Madison, Wis.), as well as otherliposomes developed according to procedures standard in the art. Inaddition, the disclosed nucleic acid or vector can be delivered in vivoby electroporation, the technology for which is available fromGenetronics, Inc. (San Diego, Calif.) as well as by means of aSONOPORATION machine (ImaRx Pharmaceutical Corp., Tucson, Ariz.).

As one example, vector delivery can be via a viral system, such as aretroviral vector system which can package a recombinant retroviralgenome (see e.g., Pastan et al., Proc. Natl. Acad. Sci. U.S.A. 85:4486,1988; Miller et al., Mol. Cell. Biol. 6:2895, 1986). The recombinantretrovirus can then be used to infect and thereby deliver to theinfected cells nucleic acid encoding a broadly neutralizing antibody (oractive fragment thereof). The exact method of introducing the alterednucleic acid into mammalian cells is, of course, not limited to the useof retroviral vectors. Other techniques are widely available for thisprocedure including the use of adenoviral vectors (Milani et al., Hum.Gene Ther. 5:941-948, 1994), adeno-associated viral (AAV) vectors(Goodman et al., Blood 84:1492-1500, 1994), lentiviral vectors (Naidiniet al., Science 272:263-267, 1996), pseudotyped retroviral vectors(Agrawal et al., Exper. Hematol. 24:738-747, 1996). Physicaltransduction techniques can also be used, such as liposome delivery andreceptor-mediated and other endocytosis mechanisms (see, for example,Schwartzenberger et al., Blood 87:472-478, 1996). This disclosedcompositions and methods can be used in conjunction with any of these orother commonly used gene transfer methods.

As one example, if the antibody-encoding nucleic acid is delivered tothe cells of a subject in an adenovirus vector, the dosage foradministration of adenovirus to humans can range from about 10⁷ to 10⁹plaque forming units (pfu) per injection but can be as high as 10¹² pfuper injection (Crystal, Hum. Gene Ther. 8:985-1001, 1997; Alvarez andCuriel, Hum. Gene Ther. 8:597-613, 1997). A subject can receive a singleinjection, or, if additional injections are necessary, they can berepeated at six month intervals (or other appropriate time intervals, asdetermined by the skilled practitioner) for an indefinite period and/oruntil the efficacy of the treatment has been established.

Parenteral administration of the nucleic acid or vector, if used, isgenerally characterized by injection. Injectables can be prepared inconventional forms, either as liquid solutions or suspensions, solidforms suitable for solution of suspension in liquid prior to injection,or as emulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. For additionaldiscussion of suitable formulations and various routes of administrationof therapeutic compounds, see, e.g., Remington: The Science and Practiceof Pharmacy (19th ed.) ed. A. R. Gennaro, Mack Publishing Company,Easton, Pa. 1995.

There are a number of compositions and methods which can be used todeliver nucleic acids to cells, either in vitro or in vivo. Thesemethods and compositions can largely be broken down into two classes:viral based delivery systems and non-viral based delivery systems. Forexample, the nucleic acids can be delivered through a number of directdelivery systems such as, electroporation, lipofection, calciumphosphate precipitation, plasmids, viral vectors, viral nucleic acids,phage nucleic acids, phages, cosmids, or via transfer of geneticmaterial in cells or carriers such as cationic liposomes. Appropriatemeans for transfection, including viral vectors, chemical transfectants,or physico-mechanical methods such as electroporation and directdiffusion of DNA, are described by, for example, Wolff, J. A., et al.,Science, 247, 1465-1468, (1990); and Wolff, J. A. Nature, 352, 815-818,(1991) Such methods are well known in the art and readily adaptable foruse with the compositions and methods described herein. In certaincases, the methods will be modified to specifically function with largeDNA molecules. Further, these methods can be used to target certaindiseases and cell populations by using the targeting characteristics ofthe carrier.

Transfer vectors can be any nucleotide construction used to delivergenes into cells (e.g., a plasmid), or as part of a general strategy todeliver genes, e.g., as part of recombinant retrovirus or adenovirus(Ram et al. Cancer Res. 53:83-88, (1993)).

As used herein, plasmid or viral vectors are agents that transport thedisclosed nucleic acids, such as a given haplotype of FVIII into thecell without degradation, and include a promoter yielding expression ofthe gene in the cells into which it is delivered. Viral vectors are, forexample, Adenovirus, Adeno-associated virus, Herpes virus, Vacciniavirus, Polio virus, AIDS virus, neuronal trophic virus, Sindbis andother RNA viruses, including these viruses with the HIV backbone. Alsopreferred are any viral families which share the properties of theseviruses which make them suitable for use as vectors. Retrovirusesinclude Murine Maloney Leukemia virus, MMLV, and retroviruses thatexpress the desirable properties of MMLV as a vector. Retroviral vectorsare able to carry a larger genetic payload, i.e., a transgene or markergene, than other viral vectors, and for this reason are a commonly usedvector. However, they are not as useful in non-proliferating cells.Adenovirus vectors are relatively stable and easy to work with, havehigh titers, and can be delivered in aerosol formulation, and cantransfect non-dividing cells. Pox viral vectors are large and haveseveral sites for inserting genes, they are thermostable and can bestored at room temperature. A preferred embodiment is a viral vectorwhich has been engineered so as to suppress the immune response of thehost organism, elicited by the viral antigens. Preferred vectors of thistype will carry coding regions for Interleukin 8 or 10.

Viral vectors can have higher transaction (ability to introduce genes)abilities than chemical or physical methods to introduce genes intocells. Typically, viral vectors contain, nonstructural early genes,structural late genes, an RNA polymerase DI transcript, invertedterminal repeats necessary for replication and encapsidation, andpromoters to control the transcription and replication of the viralgenome. When engineered as vectors, viruses typically have one or moreof the early genes removed and a gene or gene/promotor cassette isinserted into the viral genome in place of the removed viral DNA.Constructs of this type can carry up to about 8 kb of foreign geneticmaterial. The necessary functions of the removed early genes aretypically supplied by cell lines which have been engineered to expressthe gene products of the early genes in trans.

(1) Retroviral Vectors

A retrovirus is an animal virus belonging to the virus family ofRetroviridae, including any types, subfamilies, genus, or tropisms.Retroviral vectors, in general, are described by Verma, I. M.,Retroviral vectors for gene transfer. In Microbiology-1985, AmericanSociety for Microbiology, pp. 229-232, Washington, (1985), which isincorporated by reference herein. Examples of methods for usingretroviral vectors for gene therapy are described in U.S. Pat. Nos.4,868,116 and 4,980,286; PCT applications WO 90/02806 and WO 89/07136;and Mulligan, (Science 260:926-932 (1993)); the teachings of which areincorporated herein by reference.

A retrovirus is essentially a package which has packed into it nucleicacid cargo. The nucleic acid cargo carries with it a packaging signal,which ensures that the replicated daughter molecules will be efficientlypackaged within the package coat. In addition to the package signal,there are a number of molecules which are needed in cis, for thereplication, and packaging of the replicated virus. Typically aretroviral genome, contains the gag, pol, and env genes which areinvolved in the making of the protein coat. It is the gag, pol, and envgenes which are typically replaced by the foreign DNA that it is to betransferred to the target cell. Retrovirus vectors typically contain apackaging signal for incorporation into the package coat, a sequencewhich signals the start of the gag transcription unit, elementsnecessary for reverse transcription, including a primer binding site tobind the tRNA primer of reverse transcription, terminal repeat sequencesthat guide the switch of RNA strands during DNA synthesis, a purine richsequence 5′ to the 3′ LTR that serve as the priming site for thesynthesis of the second strand of DNA synthesis, and specific sequencesnear the ends of the LTRs that enable the insertion of the DNA state ofthe retrovirus to insert into the host genome. The removal of the gag,pol, and env genes allows for about 8 kb of foreign sequence to beinserted into the viral genome, become reverse transcribed, and uponreplication be packaged into a new retroviral particle. This amount ofnucleic acid is sufficient for the delivery of a one to many genesdepending on the size of each transcript. It is preferable to includeeither positive or negative selectable markers along with other genes inthe insert.

Since the replication machinery and packaging proteins in mostretroviral vectors have been removed (gag, pol, and env), the vectorsare typically generated by placing them into a packaging cell line. Apackaging cell line is a cell line which has been transfected ortransformed with a retrovirus that contains the replication andpackaging machinery, but lacks any packaging signal. When the vectorcarrying the DNA of choice is transfected into these cell lines, thevector containing the gene of interest is replicated and packaged intonew retroviral particles, by the machinery provided in cis by the helpercell. The genomes for the machinery are not packaged because they lackthe necessary signals.

(2) Adenoviral Vectors

The construction of replication-defective adenoviruses has beendescribed (Berkner et al., J. Virology 61:1213-1220 (1987); Massie etal., Mol. Cell. Biol. 6:2872-2883 (1986); Haj-Ahmad et al., J. Virology57:267-274 (1986); Davidson et al., J. Virology 61:1226-1239 (1987);Zhang “Generation and identification of recombinant adenovirus byliposome-mediated transfection and PCR analysis” BioTechniques15:868-872 (1993)). The benefit of the use of these viruses as vectorsis that they are limited in the extent to which they can spread to othercell types, since they can replicate within an initial infected cell,but are unable to form new infectious viral particles. Recombinantadenoviruses have been shown to achieve high efficiency gene transferafter direct, in vivo delivery to airway epithelium, hepatocytes,vascular endothelium, CNS parenchyma and a number of other tissue sites(Morsy, J. Clin. Invest. 92:1580-1586 (1993); Kirshenbaum, J. Clin.Invest. 92:381-387 (1993); Roessler, J. Clin. Invest. 92:1085-1092(1993); Moullier, Nature Genetics 4:154-159 (1993); La Salle, Science259:988-990 (1993); Gomez-Foix, J. Biol. Chem. 267:25129-25134 (1992);Rich, Human Gene Therapy 4:461-476 (1993); Zabner, Nature Genetics6:75-83 (1994); Guzman, Circulation Research 73:1201-1207 (1993); Bout,Human Gene Therapy 5:3-10 (1994); Zabner, Cell 75:207-216 (1993);Caillaud, Eur. J. Neuroscience 5:1287-1291 (1993); and Ragot, J. Gen.Virology 74:501-507 (1993)). Recombinant adenoviruses achieve genetransduction by binding to specific cell surface receptors, after whichthe virus is internalized by receptor-mediated endocytosis, in the samemanner as wild type or replication-defective adenovirus (Chardonnet andDales, Virology 40:462-477 (1970); Brown and Burlingham, J. Virology12:386-396 (1973); Svensson and Persson, J. Virology 55:442-449 (1985);Seth, et al., J. Virol. 51:650-655 (1984); Seth, et al., Mol. Cell.Biol. 4:1528-1533 (1984); Varga et al., J. Virology 65:6061-6070 (1991);Wickham et al., Cell 73:309-319 (1993)).

A viral vector can be one based on an adenovirus which has had the E1gene removed and these virons are generated in a cell line such as thehuman 293 cell line. In another preferred embodiment both the E1 and E3genes are removed from the adenovirus genome.

(3) Adeno-Asscociated Viral Vectors

Another type of viral vector is based on an adeno-associated virus(AAV). This defective parvovirus is a preferred vector because it caninfect many cell types and is nonpathogenic to humans. AAV type vectorscan transport about 4 to 5 kb and wild type AAV is known to stablyinsert into chromosome 19. Vectors which contain this site specificintegration property are preferred. An especially preferred embodimentof this type of vector is the P4.1 C vector produced by Avigen, SanFrancisco, Calif., which can contain the herpes simplex virus thymidinekinase gene, HSV-tk, and/or a marker gene, such as the gene encoding thegreen fluorescent protein, GFP.

In another type of AAV virus, the AAV contains a pair of invertedterminal repeats (ITRs) which flank at least one cassette containing apromoter which directs cell-specific expression operably linked to aheterologous gene. Heterologous in this context refers to any nucleotidesequence or gene which is not native to the AAV or B19 parvovirus.

Typically the AAV and B19 coding regions have been deleted, resulting ina safe, noncytotoxic vector. The AAV ITRs, or modifications thereofconfer infectivity and site-specific integration, but not cytotoxicity,and the promoter directs cell-specific expression. U.S. Pat. No.6,261,834 is herein incorproated by reference for material related tothe AAV vector.

The disclosed vectors thus provide DNA molecules which are capable ofintegration into a mammalian chromosome without substantial toxicity.

The inserted genes in viral and retroviral usually contain promoters,and/or enhancers to help control the expression of the desired geneproduct. A promoter is generally a sequence or sequences of DNA thatfunction when in a relatively fixed location in regard to thetranscription start site. A promoter contains core elements required forbasic interaction of RNA polymerase and transcription factors, and maycontain upstream elements and response elements.

(4) Large Payload Viral Vectors

Molecular genetic experiments with large human herpesviruses haveprovided a means whereby large heterologous DNA fragments can be cloned,propagated and established in cells permissive for infection withherpesviruses (Sun et al., Nature is genetics 8: 33-41, 1994; Cotter andRobertson, Curr Opin Mol Ther 5: 633-644, 1999). These large DNA viruses(herpes simplex virus (HSV) and Epstein-Barr virus (EBV), have thepotential to deliver fragments of human heterologous DNA>150 kb tospecific cells. EBV recombinants can maintain large pieces of DNA in theinfected B-cells as episomal DNA. Individual clones carried humangenomic inserts up to 330 kb appeared genetically stable The maintenanceof these episomes requires a specific EBV nuclear protein, EBNA1,constitutively expressed during infection with EBV. Additionally, thesevectors can be used for transfection, where large amounts of protein canbe generated transiently in vitro. Herpesvirus amplicon systems are alsobeing used to package pieces of DNA>220 kb and to infect cells that canstably maintain DNA as episomes.

Other useful systems include, for example, replicating andhost-restricted non-replicating vaccinia virus vectors.

e) Non-Nucleic Acid Based Systems

The disclosed compositions can be delivered to the target cells in avariety of ways. For example, the compositions can be delivered throughelectroporation, or through lipofection, or through calcium phosphateprecipitation. The delivery mechanism chosen will depend in part on thetype of cell targeted and whether the delivery is occurring for examplein vivo or in vitro.

Thus, the compositions can comprise, in addition to the disclosed genesor vectors for example, lipids such as liposomes, such as cationicliposomes (e.g., DOTMA, DOPE, DC-cholesterol) or anionic liposomes.Liposomes can further comprise proteins to facilitate targeting aparticular cell, if desired. Administration of a composition comprisinga compound and a cationic liposome can be administered to the bloodafferent to a target organ or inhaled into the respiratory tract totarget cells of the respiratory tract. Regarding liposomes, see, e.g.,Brigham et al. Am. J. Resp. Cell. Mol. Biol. 1:95-100 (1989); Feigner etal. Proc. Natl. Acad. Sci USA 84:7413-7417 (1987); U.S. Pat. No.4,897,355. Furthermore, the compound can be administered as a componentof a microcapsule that can be targeted to specific cell types, such asmacrophages, or where the diffusion of the compound or delivery of thecompound from the microcapsule is designed for a specific rate ordosage.

In the methods described above which include the administration anduptake of exogenous DNA into the cells of a subject (i.e., genetransduction or transfection), delivery of the compositions to cells canbe via a variety of mechanisms. As one example, delivery can be via aliposome, using commercially available liposome preparations such asLIPOFECTIN, LIPOFECTAMINE (GIBCO-BRL, Inc., Gaithersburg, Md.),SUPERFECT (Qiagen, Inc. Hilden, Germany) and TRANSFECTAM (PromegaBiotec, Inc., Madison, Wis.), as well as other liposomes developedaccording to procedures standard in the art. In addition, the disclosednucleic acid or vector can be delivered in vivo by electroporation, thetechnology for which is available from Genetronics, Inc. (San Diego,Calif.) as well as by means of a SONOPORATION machine (ImaRxPharmaceutical Corp., Tucson, Ariz.).

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al., BioconjugateChem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., CancerImmunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). These techniques can be used for avariety of other speciifc cell types. Vehicles such as “stealth” andother antibody conjugated liposomes (including lipid mediated drugtargeting to colonic carcinoma), receptor mediated targeting of DNAthrough cell specific ligands, lymphocyte directed tumor targeting, andhighly specific therapeutic retroviral targeting of murine glioma cellsin vivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

Nucleic acids that are delivered to cells which are to be integratedinto the host cell genome, typically contain integration sequences.These sequences are often viral related sequences, particularly whenviral based systems are used. These viral intergration systems can alsobe incorporated into nucleic acids which are to be delivered using anon-nucleic acid based system of deliver, such as a liposome, so thatthe nucleic acid contained in the delivery, system can be comeintegrated into the host genome.

Other general techniques for integration into the host genome include,for example, systems designed to promote homologous recombination withthe host genome. These systems typically rely on sequence flanking thenucleic acid to be expressed that has enough homology with a targetsequence within the host cell genome that recombination between thevector nucleic acid and the target nucleic acid takes place, causing thedelivered nucleic acid to be integrated into the host genome. Thesesystems and the methods necessary to promote homologous recombinationare known to those of skill in the art.

f) In Vivo/Ex Vivo

As described above, the compositions can be administered in apharmaceutically acceptable carrier and can be delivered to thesubject=s cells in vivo and/or ex vivo by a variety of mechanisms wellknown in the art (e.g., uptake of naked DNA, liposome fusion,intramuscular injection of DNA via a gene gun, endocytosis and thelike).

If ex vivo methods are employed, cells or tissues can be removed andmaintained outside the body according to standard protocols well knownin the art. The compositions can be introduced into the cells via anygene transfer mechanism, such as, for example, calcium phosphatemediated gene delivery, electroporation, microinjection orproteoliposomes. The transduced cells can then be infused (e.g., in apharmaceutically acceptable carrier) or homotopically transplanted backinto the subject per standard methods for the cell or tissue type.Standard methods are known for transplantation or infusion of variouscells into a subject.

4. Expression Systems

The nucleic acids that are delivered to cells typically containexpression controlling systems. For example, the inserted genes in viraland retroviral systems usually contain promoters, and/or enhancers tohelp control the expression of the desired gene product. A promoter isgenerally a sequence or sequences of DNA that function when in arelatively fixed location in regard to the transcription start site. Apromoter contains core elements required for basic interaction of RNApolymerase and transcription factors, and may contain upstream elementsand response elements.

a) Viral Promoters and Enhancers

Preferred promoters controlling transcription from vectors in mammalianhost cells may be obtained from various sources, for example, thegenomes of viruses such as: polyoma, Simian Virus 40 (SV40), adenovirus,retroviruses, hepatitis-B virus and most preferably cytomegalovirus, orfrom heterologous mammalian promoters, e.g. beta actin promoter. Theearly and late promoters of the SV40 virus are conveniently obtained asan SV40 restriction fragment which also contains the SV40 viral originof replication (Fiers et al., Nature, 273: 113 (1978)). The immediateearly promoter of the human cytomegalovirus is conveniently obtained asa HindIII E restriction fragment (Greenway, P. J. et al., Gene 18:355-360 (1982)). Of course, promoters from the host cell or relatedspecies also are useful herein.

Enhancer generally refers to a sequence of DNA that functions at nofixed distance from the transcription start site and can be either 5′(Laimins, L. et al., Proc. Natl. Acad. Sci. 78: 993 (1981)) or 3′(Lusky, M. L., et al., Mol. Cell Bio. 3: 1108 (1983)) to thetranscription unit. Furthermore, enhancers can be within an intron(Banerji, J. L. et al., Cell 33: 729 (1983)) as well as within thecoding sequence itself (Osborne, T. F., et al., Mol. Cell Bio. 4: 1293(1984)). They are usually between 10 and 300 by in length, and theyfunction in cis. Enhancers function to increase transcription fromnearby promoters. Enhancers also often contain response elements thatmediate the regulation of transcription. Promoters can also containresponse elements that mediate the regulation of transcription.Enhancers often determine the regulation of expression of a gene. Whilemany enhancer sequences are now known from mammalian genes (globin,elastase, albumin, -fetoprotein and insulin), typically one will use anenhancer from a eukaryotic cell virus for general expression. Preferredexamples are the SV40 enhancer on the late side of the replicationorigin (bp 100-270), the cytomegalovirus early promoter enhancer, thepolyoma enhancer on the late side of the replication origin, andadenovirus enhancers.

The promotor and/or enhancer may be specifically activated either bylight or specific chemical events which trigger their function. Systemscan be regulated by reagents such as tetracycline and dexamethasone.There are also ways to enhance viral vector gene expression by exposureto irradiation, such as gamma irradiation, or alkylating chemotherapydrugs.

In certain embodiments the promoter and/or enhancer region can act as aconstitutive promoter and/or enhancer to maximize expression of theregion of the transcription unit to be transcribed. In certainconstructs the promoter and/or enhancer region be active in alleukaryotic cell types, even if it is only expressed in a particular typeof cell at a particular time. A preferred promoter of this type is theCMV promoter (650 bases). Other preferred promoters are SV40 promoters,cytomegalovirus (full length promoter), and retroviral vector LTR.

It has been shown that all specific regulatory elements can be clonedand used to construct expression vectors that are selectively expressedin specific cell types such as melanoma cells. The glial fibrillaryacetic protein (GFAP) promoter has been used to selectively expressgenes in cells of glial origin.

Expression vectors used in eukaryotic host cells (yeast, fungi, insect,plant, animal, human or nucleated cells) may also contain sequencesnecessary for the termination of transcription which may affect mRNAexpression. These regions are transcribed as polyadenylated segments inthe untranslated portion of the mRNA encoding tissue factor protein. The3′ untranslated regions also include transcription termination sites. Itis preferred that the transcription unit also contain a polyadenylationregion. One benefit of this region is that it increases the likelihoodthat the transcribed unit will be processed and transported like mRNA.The identification and use of polyadenylation signals in expressionconstructs is well established. It is preferred that homologouspolyadenylation signals be used in the transgene constructs. In certaintranscription units, the polyadenylation region is derived from the SV40early polyadenylation signal and consists of about 400 bases. It is alsopreferred that the transcribed units contain other standard sequencesalone or in combination with the above sequences improve expressionfrom, or stability of, the construct.

b) Markers

The viral vectors can include nucleic acid sequence encoding a markerproduct. This marker product is used to determine if the gene has beendelivered to the cell and once delivered is being expressed. Preferredmarker genes are the E. Coli lacZ gene, which encodes β-galactosidase,and green fluorescent protein.

In some embodiments the marker may be a selectable marker. Examples ofsuitable selectable markers for mammalian cells are dihydrofolatereductase (DHFR), thymidine kinase, neomycin, neomycin analog G418,hydromycin, and puromycin. When such selectable markers are successfullytransferred into a mammalian host cell, the transformed mammalian hostcell can survive if placed under selective pressure. There are twowidely used distinct categories of selective regimes. The first categoryis based on a cell's metabolism and the use of a mutant cell line whichlacks the ability to grow independent of a supplemented media. Twoexamples are: CHO DHFR-cells and mouse LTK-cells. These cells lack theability to grow without the addition of such nutrients as thymidine orhypoxanthine. Because these cells lack certain genes necessary for acomplete nucleotide synthesis pathway, they cannot survive unless themissing nucleotides are provided in a supplemented media. An alternativeto supplementing the media is to introduce an intact DHFR or TK geneinto cells lacking the respective genes, thus altering their growthrequirements. Individual cells which were not transformed with the DHFRor TK gene will not be capable of survival in non-supplemented media.

The second category is dominant selection which refers to a selectionscheme used in any cell type and does not require the use of a mutantcell line. These schemes typically use a drug to arrest growth of a hostcell. Those cells which have a novel gene would express a proteinconveying drug resistance and would survive the selection. Examples ofsuch dominant selection use the drugs neomycin, (Southern P. and Berg,P., J. Molec. Appl. Genet. 1: 327 (1982)), mycophenolic acid, (Mulligan,R. C. and Berg, P. Science 209: 1422 (1980)) or hygromycin, (Sugden, B.et al., Mol. Cell. Biol. 5: 410-413 (1985)). The three examples employbacterial genes under eukaryotic control to convey resistance to theappropriate drug G418 or neomycin (geneticin), xgpt (mycophenolic acid)or hygromycin, respectively. Others include the neomycin analog G418 andpuramycin.

5. Peptides

a) Protein Variants

As discussed herein there are numerous variants of the FVIII proteinthat are disclosed. As described above, the present invention providesat least four common non-synonymous-single-nucleotide-polymorphisms(nsSNPs), combinations of which represent six naturally-occurringallelic variants of the FVIII protein in the human population (FIG. 2).As such, six different haplotypic forms of the wt FVIII protein havebeen identified. These haplotypic forms have been designated: H1, H2,H3, H4, H5, and H6 (FIG. 3). Each of these variants represents a normalallelic variant of the FVIII protein since the individuals from whom thesequences were described have no bleeding disorders.

Protein variation, as described herein, applies generally to the design,synthesis, and recogniztion of proteins. However, the present inventionis drawn to specific haplotypes with specific nucleic acid, and aminoacid, sequences. These sequences need not be varied as disclosed herein,but can optionally contain other variances than those disclosed inhaplotypes H1-H6.

Amino acid sequence modifications typically fall into one or more ofthree classes: substitutional, insertional or deletional variants.Insertions include amino and/or carboxyl terminal fusions as well asintrasequence insertions of single or multiple amino acid residues.Insertions ordinarily will be smaller insertions than those of amino orcarboxyl terminal fusions, for example, on the order of one to fourresidues. Immunogenic fusion protein derivatives, such as thosedescribed in the examples, are made by fusing a polypeptide sufficientlylarge to confer immunogenicity to the target sequence by cross-linkingin vitro or by recombinant cell culture transformed with DNA encodingthe fusion. Deletions are characterized by the removal of one or moreamino acid residues from the protein sequence. Typically, no more thanabout from 2 to 6 residues are deleted at any one site within theprotein molecule. These variants ordinarily are prepared by sitespecific mutagenesis of nucleotides in the DNA encoding the protein,thereby producing DNA encoding the variant, and thereafter expressingthe DNA in recombinant cell culture. Techniques for making substitutionmutations at predetermined sites in DNA having a known sequence are wellknown, for example M13 primer mutagenesis and PCR mutagenesis. Aminoacid substitutions are typically of single residues, but can occur at anumber of different locations at once; insertions usually will be on theorder of about from 1 to 10 amino acid residues; and deletions willrange about from 1 to 30 residues. Deletions or insertions preferablyare made in adjacent pairs, i.e. a deletion of 2 residues or insertionof 2 residues. Substitutions, deletions, insertions or any combinationthereof may be combined to arrive at a final construct. The mutationsmust not place the sequence out of reading frame and preferably will notcreate complementary regions that could produce secondary mRNAstructure. Substitutional variants are those in which at least oneresidue has been removed and a different residue inserted in its place.Such substitutions generally are made in accordance with the followingTables AAA and AAS and are referred to as conservative substitutions.

TABLE AAA Amino Acid Abbreviations Amino Acid Abbreviations alanine Ala,A alloisoleucine aIle arginine Arg, R asparagine Asn, N aspartic acidAsp, D cysteine Cys, C glutamic acid Glu, E glutamine Gln, K glycineGly, G histidine His, H isoleucine Ile, I leucine Leu, L lysine Lys, Kphenylalanine Phe, F proline Pro, P pyroglutamic acid pGlu serine Ser, Sthreonine Thr, T tyrosine Tyr, Y tryptophan Trp, W valine Val, V

TABLE AAS Amino Acid Substitutions Exemplary Conservative Substitutions,Original Residue others are known in the art. Ala Ser Arg Lys; Gln AsnGln; His Asp Glu Cys Ser Gln Asn; Lys Glu Asp Gly Pro His Asn; Gln IleLeu; Val Leu Ile; Val Lys Arg; Gln Met Leu; Ile Phe Met; Leu; Tyr SerThr Thr Ser Trp Tyr Tyr Trp; Phe Val Ile; Leu

Substantial changes in function or immunological identity are made byselecting substitutions that are less conservative than those in TableAAS, i.e., selecting residues that differ more significantly in theireffect on maintaining (a) the structure of the polypeptide backbone inthe area of the substitution, for example as a sheet or helicalconformation, (b) the charge or hydrophobicity of the molecule at thetarget site or (c) the bulk of the side chain. The substitutions whichin general are expected to produce the greatest changes in the proteinproperties will be those in which (a) a hydrophilic residue, e.g. serylor threonyl, is substituted for (or by) a hydrophobic residue, e.g.leucyl, isoleucyl, phenylalanyl, valyl or alanyl; (b) a cysteine orproline is substituted for (or by) any other residue; (c) a residuehaving an electropositive side chain, e.g., lysyl, arginyl, or histidyl,is substituted for (or by) an electronegative residue, e.g., glutamyl oraspartyl; or (d) a residue having a bulky side chain, e.g.,phenylalanine, is substituted for (or by) one not having a side chain,e.g., glycine, in this case, (e) by increasing the number of sites forsulfation and/or glycosylation.

For example, the replacement of one amino acid residue with another thatis biologically and/or chemically similar is known to those skilled inthe art as a conservative substitution. For example, a conservativesubstitution would be replacing one hydrophobic residue for another, orone polar residue for another. The substitutions include combinationssuch as, for example, Gly, Ala; Val, Ile, Leu; Asp, Glu; Asn, Gln; Ser,Thr; Lys, Arg; and Phe, Tyr. Such conservatively substituted variationsof each explicitly disclosed sequence are included within the mosaicpolypeptides provided herein.

Substitutional or deletional mutagenesis can be employed to insert sitesfor N-glycosylation (Asn-X-Thr/Ser) or O-glycosylation (Ser or Thr).Deletions of cysteine or other labile residues also may be desirable.Deletions or substitutions of potential proteolysis sites, e.g. Mg, isaccomplished for example by deleting one of the basic residues orsubstituting one by glutaminyl or histidyl residues.

Certain post-translational derivatizations are the result of the actionof recombinant host cells on the expressed polypeptide. Glutaminyl andasparaginyl residues are frequently post-translationally deamidated tothe corresponding glutamyl and asparyl residues. Alternatively, theseresidues are deamidated under mildly acidic conditions. Otherpost-translational modifications include hydroxylation of proline andlysine, phosphorylation of hydroxyl groups of seryl or threonylresidues, methylation of the o-amino groups of lysine, arginine, andhistidine side chains (T. E. Creighton, Proteins: Structure andMolecular Properties, W. H. Freeman & Co., San Francisco pp 79-86[1983]), acetylation of the N-terminal amine and, in some instances,amidation of the C-terminal carboxyl.

It is understood that one way to define the variants and derivatives ofthe disclosed proteins herein is through defining the variants andderivatives in terms of homology/identity to specific known sequences.Specifically disclosed are variants of these and other proteins hereindisclosed which have at least, 70% or 75% or 80% or 85% or 90% or 95%homology to the stated sequence. Those of skill in the art readilyunderstand how to determine the homology of two proteins. For example,the homology can be calculated after aligning the two sequences so thatthe homology is at its highest level.

Another way of calculating homology can be performed by publishedalgorithms. Optimal alignment of sequences for comparison may beconducted by the local homology algorithm of Smith and Waterman Adv.Appl. Math. 2: 482 (1981), by the homology alignment algorithm ofNeedleman and Wunsch, J. Mol. Biol. 48: 443 (1970), by the search forsimilarity method of Pearson and Lipman, Proc. Natl. Acad. Sci. U.S.A.85: 2444 (1988), by computerized implementations of these algorithms(GAP, BESTFIT, FASTA, and TFASTA in the Wisconsin Genetics SoftwarePackage, Genetics Computer Group, 575 Science Dr., Madison, Wis.), or byinspection.

The same types of homology can be obtained for nucleic acids by forexample the algorithms disclosed in Zuker, M. Science 244:48-52, 1989,Jaeger et al. Proc. Natl. Acad. Sci. USA 86:7706-7710, 1989, Jaeger etal. Methods Enzymol. 183:281-306, 1989 which are herein incorporated byreference for at least material related to nucleic acid alignment.

It is understood that the description of conservative mutations andhomology can be combined together in any combination, such asembodiments that have at least 70% homology to a particular sequencewherein the variants are conservative mutations.

As this specification discusses various proteins and protein sequencesit is understood that the nucleic acids that can encode those proteinsequences are also disclosed. This would include all degeneratesequences related to a specific protein sequence, i.e. all nucleic acidshaving a sequence that encodes one particular protein sequence as wellas all nucleic acids, including degenerate nucleic acids, encoding thedisclosed variants and derivatives of the protein sequences. Thus, whileeach particular nucleic acid sequence may not be written out herein, itis understood that each and every sequence is in fact disclosed anddescribed herein through the disclosed protein sequence. It is alsounderstood that while no amino acid sequence indicates what particularDNA sequence encodes that protein within an organism, where particularvariants of a disclosed protein are disclosed herein, the known nucleicacid sequence that encodes that protein in the particular gene fromwhich that protein arises is also known and herein disclosed anddescribed.

It is understood that there are numerous amino acid and peptide analogswhich can be incorporated into the disclosed compositions. For example,there are numerous D amino acids or amino acids which have a differentfunctional substituent then the amino acids shown in Table AAA and TableAAS. The opposite stereo isomers of naturally occurring peptides aredisclosed, as well as the stereo isomers of peptide analogs. These aminoacids can readily be incorporated into polypeptide chains by chargingtRNA molecules with the amino acid of choice and engineering geneticconstructs that utilize, for example, amber codons, to insert the analogamino acid into a peptide chain in a site specific way (Thorson et al.,Methods in Molec. Biol. 77:43-73 (1991), Zoller, Current Opinion inBiotechnology, 3:348-354 (1992); Ibba, Biotechnology & GeneticEnginerring Reviews 13:197-216 (1995), Cahill et al., TIBS,14(10):400-403 (1989); Benner, TIB Tech, 12:158-163 (1994); Ibba andHennecke, Bio/technology, 12:678-682 (1994) all of which are hereinincorporated by reference at least for material related to amino acidanalogs).

Molecules can be produced that resemble peptides, but which are notconnected via a natural peptide linkage. For example, linkages for aminoacids or amino acid analogs can include CH₂NH—, —CH₂S—,—CH₂—CH₂—CH═CH—(cis and trans), —COCH₂—CH(OH)CH₂—, and —CHH₂SO— (Theseand others can be found in Spatola, A. F. in Chemistry and Biochemistryof Amino Acids, Peptides, and Proteins, B. Weinstein, eds., MarcelDekker, New York, p. 267 (1983); Spatola, A. F., Vega Data (March 1983),Vol. 1, Issue 3, Peptide Backbone Modifications (general review);Morley, Trends Pharm Sci (1980) pp. 463-468; Hudson, D. et al., Int JPept Prot Res 14:177-185 (1979) (—CH₂NH—, CH₂CH₂—); Spatola et al. LifeSci 38:1243-1249 (1986) (—CHH₂—S); Hann J. Chem. Soc Perkin Trans.1307-314 (1982) (—CH—CH—, cis and trans); Almquist et al. J. Med. Chem.23:1392-1398 (1980) (—COCH₂—); Jennings-White et al. Tetrahedron Lett23:2533 (1982) (—COCH₂—); Szelke et al. European Appin, EP 45665 CA(1982): 97:39405 (1982) (—CH(OH)CH₂—); Holladay et al. Tetrahedron. Lett24:4401-4404 (1983) (—C(OH)CH₂—); and Hruby Life Sci 31:189-199 (1982)(—CH₂—S—); each of which is incorporated herein by reference. Aparticularly preferred non-peptide linkage is —CH₂NH—. It is understoodthat peptide analogs can have more than one atom between the bond atoms,such as b-alanine, g-aminobutyric acid, and the like.

Amino acid analogs and analogs and peptide analogs often have enhancedor desirable properties, such as, more economical production, greaterchemical stability, enhanced pharmacological properties (half-life,absorption, potency, efficacy, etc.), altered specificity (e.g., abroad-spectrum of biological activities), reduced antigenicity, andothers.

D-amino acids can be used to generate more stable peptides, because Damino acids are not recognized by peptidases and such. Systematicsubstitution of one or more amino acids of a consensus sequence with aD-amino acid of the same type (e.g., D-lysine in place of L-lysine) canbe used to generate more stable peptides. Cysteine residues can be usedto cyclize or attach two or more peptides together. This can bebeneficial to constrain peptides into particular conformations. (Rizoand Gierasch Ann. Rev. Biochem. 61:387 (1992), incorporated herein byreference).

6. Antibodies

(1) Antibodies Generally

The term “antibodies” is used herein in a broad sense and includes bothpolyclonal and monoclonal antibodies. In addition to intactimmunoglobulin molecules, also included in the term “antibodies” arefragments or polymers of those immunoglobulin molecules, and human orhumanized versions of immunoglobulin molecules or fragments thereof, aslong as they are chosen for their ability to interact with a specifichaplotype of FVIII such that FVIII is inhibited, for example. Theantibodies can be tested for their desired activity using the in vitroassays described herein, or by analogous methods, after which their invivo therapeutic and/or prophylactic activities are tested according toknown clinical testing methods.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a substantially homogeneous population of antibodies,i.e., the individual antibodies within the population are identicalexcept for possible naturally occurring mutations that may be present ina small subset of the antibody molecules. The monoclonal antibodiesherein specifically include “chimeric” antibodies in which a portion ofthe heavy and/or light chain is identical with or homologous tocorresponding sequences in antibodies derived from a particular speciesor belonging to a particular antibody class or subclass, while theremainder of the chain(s) is identical with or homologous tocorresponding sequences in antibodies derived from another species orbelonging to another antibody class or subclass, as well as fragments ofsuch antibodies, as long as they exhibit the desired antagonisticactivity (See, U.S. Pat. No. 4,816,567 and Morrison et al., Proc. Natl.Acad. Sci. USA, 81:6851-6855 (1984)).

The disclosed monoclonal antibodies can be made using any procedurewhich produces monoclonal antibodies. For example, disclosed monoclonalantibodies can be prepared using hybridoma methods, such as thosedescribed by Kohler and Milstein, Nature, 256:495 (1975). In a hybridomamethod, a mouse or other appropriate host animal is typically immunizedwith an immunizing agent to elicit lymphocytes that produce or arecapable of producing antibodies that will specifically bind to theimmunizing agent. Alternatively, the lymphocytes may be immunized invitro, e.g., using the HIV Env-CD4-co-receptor complexes describedherein.

The monoclonal antibodies may also be made by recombinant DNA methods,such as those described in U.S. Pat. No. 4,816,567 (Cabilly et al.). DNAencoding the disclosed monoclonal antibodies can be readily isolated andsequenced using conventional procedures (e.g., by using oligonucleotideprobes that are capable of binding specifically to genes encoding theheavy and light chains of murine antibodies). Libraries of antibodies oractive antibody fragments can also be generated and screened using phagedisplay techniques, e.g., as described in U.S. Pat. No. 5,804,440 toBurton et al. and U.S. Pat. No. 6,096,441 to Barbas et al.

In vitro methods are also suitable for preparing monovalent antibodies.Digestion of antibodies to produce fragments thereof, particularly, Fabfragments, can be accomplished using routine techniques known in theart. For instance, digestion can be performed using papain. Examples ofpapain digestion are described in WO 94/29348 published Dec. 22, 1994and U.S. Pat. No. 4,342,566. Papain digestion of antibodies typicallyproduces two identical antigen binding fragments, called Fab fragments,each with a single antigen binding site, and a residual Fc fragment.Pepsin treatment yields a fragment that has two antigen combining sitesand is still capable of cross-linking antigen.

The fragments, whether attached to other sequences or not, can alsoinclude insertions, deletions, substitutions, or other selectedmodifications of particular regions or specific amino acids residues,provided the activity of the antibody or antibody fragment is notsignificantly altered or impaired compared to the non-modified antibodyor antibody fragment. These modifications can provide for someadditional property, such as to remove/add amino acids capable ofdisulfide bonding, to increase its bio-longevity, to alter its secretorycharacteristics, etc. In any case, the antibody or antibody fragmentmust possess a bioactive property, such as-specific binding to itscognate antigen. Functional or active regions of the antibody orantibody fragment may be identified by mutagenesis of a specific regionof the protein, followed by expression and testing of the expressedpolypeptide. Such methods are readily apparent to a skilled practitionerin the art and can include site-specific mutagenesis of the nucleic acidencoding the antibody or antibody fragment. (Zoller, M. J. Curr. Opin.Biotechnol. 3:348-354, 1992).

As used herein, the term “antibody” or “antibodies” can also refer to ahuman antibody and/or a humanized antibody. Many non-human antibodies(e.g., those derived from mice, rats, or rabbits) are naturallyantigenic in humans, and thus can give rise to undesirable immuneresponses when administered to humans. Therefore, the use of human orhumanized antibodies in the methods serves to lessen the chance that anantibody administered to a human will evoke an undesirable immuneresponse.

FVIII inhibitor antibody neutralizing antibodies can be administered toneutralize endogenous FVIII inhibitor antibodies.

(2) Human Antibodies

The disclosed human antibodies can be prepared using any technique.Examples of techniques for human monoclonal antibody production includethose described by Cole et al. (Monoclonal Antibodies and CancerTherapy, Alan R. Liss, p. 77, 1985) and by Boerner et al. (J. Immunol.,147(1):86-95, 1991). Human antibodies (and fragments thereof) can alsobe produced using phage display libraries (Hoogenboom et al., J. Mol.Biol., 227:381, 1991; Marks et al., J. Mol. Biol., 222:581, 1991).

The disclosed human antibodies can also be obtained from transgenicanimals. For example, transgenic, mutant mice that are capable ofproducing a full repertoire of human antibodies, in response toimmunization, have been described (see, e.g., Jakobovits et al., Proc.Natl. Acad. Sci. USA, 90:2551-255 (1993); Jakobovits et al., Nature,362:255-258 (1993); Bruggermann et al., Year in Immunol., 7:33 (1993)).Specifically, the homozygous deletion of the antibody heavy chainjoining region (J(H)) gene in these chimeric and germ-line mutant miceresults in complete inhibition of endogenous antibody production, andthe successful transfer of the human germ-line antibody gene array intosuch germ-line mutant mice results in the production of human antibodiesupon antigen challenge. Antibodies having the desired activity areselected using Env-CD4-co-receptor complexes as described herein.

(3) Humanized Antibodies

Antibody humanization techniques generally involve the use ofrecombinant DNA technology to manipulate the DNA sequence encoding oneor more polypeptide chains of an antibody molecule. Accordingly, ahumanized form of a non-human antibody (or a fragment thereof) is achimeric antibody or antibody chain (or a fragment thereof, such as anFv, Fab, Fab′, or other antigen-binding portion of an antibody) whichcontains a portion of an antigen binding site from a non-human (donor)antibody integrated into the framework of a human (recipient) antibody.

To generate a humanized antibody, residues from one or morecomplementarity determining regions (CDRs) of a recipient (human)antibody molecule are replaced by residues from one or more CDRs of adonor (non-human) antibody molecule that is known to have desiredantigen binding characteristics (e.g., a certain level of specificityand affinity for the target antigen). In some instances, Fv framework(FR) residues of the human antibody are replaced by correspondingnon-human residues. Humanized antibodies may also contain residues whichare found neither in the recipient antibody nor in the imported CDR orframework sequences. Generally, a humanized antibody has one or moreamino acid residues introduced into it from a source which is non-human.In practice, humanized antibodies are typically human antibodies inwhich some CDR residues and possibly some FR residues are substituted byresidues from analogous sites in rodent antibodies. Humanized antibodiesgenerally contain at least a portion of an antibody constant region(Fc), typically that of a human antibody (Jones et al., Nature,321:522-525 (1986), Reichmann et al., Nature, 332:323-327 (1988), andPresta, Curr. Opin. Struct. Biol., 2:593-596 (1992)).

Methods for humanizing non-human antibodies are well known in the art.For example, humanized antibodies can be generated according to themethods of Winter and co-workers (Jones et al., Nature, 321:522-525(1986), Riechmann et al., Nature, 332:323-327 (1988), Verhoeyen et al.,Science, 239:1534-1536 (1988)), by substituting rodent CDRs or CDRsequences for the corresponding sequences of a human antibody. Methodsthat can be used to produce humanized antibodies are also described inU.S. Pat. No. 4,816,567 (Cabilly et al.), U.S. Pat. No. 5,565,332(Hoogenboom et al.), U.S. Pat. No. 5,721,367 (Kay et al.), U.S. Pat. No.5,837,243 (Deo et al.), U.S. Pat. No. 5,939,598 (Kucherlapati et al.),U.S. Pat. No. 6,130,364 (Jakobovits et al.), and U.S. Pat. No. 6,180,377(Morgan et al.).

(4) Administration of Antibodies

Administration of the antibodies can be done as disclosed herein.Nucleic acid approaches for antibody delivery also exist. FVIIIinhibitor antibody neutralizing antibodies and antibody fragments canalso be administered to patients or subjects as a nucleic acidpreparation (e.g., DNA or RNA) that encodes the antibody or antibodyfragment, such that the patient's or subject's own cells take up thenucleic acid and produce and secrete the encoded antibody or antibodyfragment. The delivery of the nucleic acid can be by any means, asdisclosed herein, for example.

7. Pharmaceutical Carriers/Delivery of Pharmaceutical Products

As described above, the compositions can also be administered in vivo ina pharmaceutically acceptable carrier. By “pharmaceutically acceptable”is meant a material that is not biologically or otherwise undesirable,i.e., the material may be administered to a subject, along with thenucleic acid or vector, without causing any undesirable biologicaleffects or interacting in a deleterious manner with any of the othercomponents of the pharmaceutical composition in which it is contained.The carrier would naturally be selected to minimize any degradation ofthe active ingredient and to minimize any adverse side effects in thesubject, as would be well known to one of skill in the art.

The compositions may be administered orally, parenterally (e.g.,intravenously), by intramuscular injection, by intraperitonealinjection, transdermally, extracorporeally, topically or the like,including topical intranasal administration or administration byinhalant. As used herein, “topical intranasal administration” meansdelivery of the compositions into the nose and nasal passages throughone or both of the nares and can comprise delivery by a sprayingmechanism or droplet mechanism, or through aerosolization of the nucleicacid or vector. Administration of the compositions by inhalant can bethrough the nose or mouth via delivery by a spraying or dropletmechanism. Delivery can also be directly to any area of the respiratorysystem (e.g., lungs) via intubation. The exact amount of thecompositions required will vary from subject to subject, depending onthe species, age, weight and general condition of the subject, theseverity of the allergic disorder being treated, the particular nucleicacid or vector used, its mode of administration and the like. Thus, itis not possible to specify an exact amount for every composition.However, an appropriate amount can be determined by one of ordinaryskill in the art using only routine experimentation given the teachingsherein.

Parenteral administration of the composition, if used, is generallycharacterized by injection. Injectables can be prepared in conventionalforms, either as liquid solutions or suspensions, solid forms suitablefor solution of suspension in liquid prior to injection, or asemulsions. A more recently revised approach for parenteraladministration involves use of a slow release or sustained releasesystem such that a constant dosage is maintained. See, e.g., U.S. Pat.No. 3,610,795, which is incorporated by reference herein.

The materials may be in solution, suspension (for example, incorporatedinto microparticles, liposomes, or cells). These may be targeted to aparticular cell type via antibodies, receptors, or receptor ligands. Thefollowing references are examples of the use of this technology totarget specific proteins to tumor tissue (Senter, et al., BioconjugateChem., 2:447-451, (1991); Bagshawe, K. D., Br. J. Cancer, 60:275-281,(1989); Bagshawe, et al., Br. J. Cancer, 58:700-703, (1988); Senter, etal., Bioconjugate Chem., 4:3-9, (1993); Battelli, et al., CancerImmunol. Immunother., 35:421-425, (1992); Pietersz and McKenzie,Immunolog. Reviews, 129:57-80, (1992); and Roffler, et al., Biochem.Pharmacol, 42:2062-2065, (1991)). Vehicles such as “stealth” and otherantibody conjugated liposomes (including lipid mediated drug targetingto colonic carcinoma), receptor mediated targeting of DNA through cellspecific ligands, lymphocyte directed tumor targeting, and highlyspecific therapeutic retroviral targeting of murine glioma cells invivo. The following references are examples of the use of thistechnology to target specific proteins to tumor tissue (Hughes et al.,Cancer Research, 49:6214-6220, (1989); and Litzinger and Huang,Biochimica et Biophysica Acta, 1104:179-187, (1992)). In general,receptors are involved in pathways of endocytosis, either constitutiveor ligand induced. These receptors cluster in clathrin-coated pits,enter the cell via clathrin-coated vesicles, pass through an acidifiedendosome in which the receptors are sorted, and then either recycle tothe cell surface, become stored intracellularly, or are degraded inlysosomes. The internalization pathways serve a variety of functions,such as nutrient uptake, removal of activated proteins, clearance ofmacromolecules, opportunistic entry of viruses and toxins, dissociationand degradation of ligand, and receptor-level regulation. Many receptorsfollow more than one intracellular pathway, depending on the cell type,receptor concentration, type of ligand, ligand valency, and ligandconcentration. Molecular and cellular mechanisms of receptor-mediatedendocytosis has been reviewed (Brown and Greene, DNA and Cell Biology10:6, 399-409 (1991)).

a) Pharmaceutically Acceptable Carriers

The compositions, including antibodies, can be used therapeutically incombination with a pharmaceutically acceptable carrier.

Suitable carriers and their formulations are described in Remington: TheScience and Practice of Pharmacy (19th ed.) ed. A. R. Gennaro, MackPublishing Company, Easton, Pa. 1995. Typically, an appropriate amountof a pharmaceutically-acceptable salt is used in the formulation torender the formulation isotonic. Examples of thepharmaceutically-acceptable carrier include, but are not limited to,saline, Ringer's solution and dextrose solution. The pH of the solutionis preferably from about 5 to about 8, and more preferably from about 7to about 7.5. Further carriers include sustained release preparationssuch as semipermeable matrices of solid hydrophobic polymers containingthe antibody, which matrices are in the form of shaped articles, e.g.,films, liposomes or microparticles. It will be apparent to those personsskilled in the art that certain carriers may be more preferabledepending upon, for instance, the route of administration andconcentration of composition being administered.

Pharmaceutical carriers are known to those skilled in the art. Thesemost typically would be standard carriers for administration of drugs tohumans, including solutions such as sterile water, saline, and bufferedsolutions at physiological pH. The compositions can be administeredintramuscularly or subcutaneously. Other compounds will be administeredaccording to standard procedures used by those skilled in the art.

Pharmaceutical compositions may include carriers, thickeners, diluents,buffers, preservatives, surface active agents and the like in additionto the molecule of choice. Pharmaceutical compositions may also includeone or more active ingredients such as antimicrobial agents,antiinflammatory agents, anesthetics, and the like.

The pharmaceutical composition may be administered in a number of waysdepending on whether local or systemic treatment is desired, and on thearea to be treated. Administration may be parenterally, for example byintravenous drip, subcutaneous, intraperitoneal or intramuscularinjection. The disclosed antibodies can be administered intravenously,intraperitoneally, intramuscularly, subcutaneouslyor intracavity.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, anti-oxidants, chelating agents, and inertgases and the like.

Some of the compositions may potentially be administered as apharmaceutically acceptable acid- or base-addition salt, formed byreaction with inorganic acids such as hydrochloric acid, hydrobromicacid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, andphosphoric acid, and organic acids such as formic acid, acetic acid,propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid,malonic acid, succinic acid, maleic acid, and fumaric acid, or byreaction with an inorganic base such as sodium hydroxide, ammoniumhydroxide, potassium hydroxide, and organic bases such as mono-, di-,trialkyl and aryl amines and substituted ethanolamines.

8. Chips and Micro Arrays

Disclosed are chips where at least one address is the sequences or partof the sequences set forth in any of the nucleic acid sequencesdisclosed herein. Also disclosed are chips where at least one address isthe sequences or portion of sequences set forth in any of the peptidesequences disclosed herein.

Also disclosed are chips where at least one address is a variant of thesequences or part of the sequences set forth in any of the nucleic acidsequences disclosed herein. Also disclosed are chips where at least oneaddress is a variant of the sequences or portion of sequences set forthin any of the peptide sequences disclosed herein.

9. Computer Readable Mediums

It is understood that the disclosed nucleic acids and proteins can berepresented as a sequence consisting of the nucleotides of amino acids.There are a variety of ways to display these sequences, for example thenucleotide guanosine can be represented by G or g. Likewise the aminoacid valine can be represented by Val or V. Those of skill in the artunderstand how to display and express any nucleic acid or proteinsequence in any of the variety of ways that exist, each of which isconsidered herein disclosed. Specifically contemplated herein is thedisplay of these sequences on computer readable mediums, such as,commercially available floppy disks, tapes, chips, hard drives, compactdisks, and video disks, or other computer readable mediums. Alsodisclosed are the binary code representations of the disclosedsequences. Those of skill in the art understand what computer readablemediums. Thus, computer readable mediums on which the nucleic acids orprotein sequences are recorded, stored, or saved.

Disclosed are computer readable mediums comprising the sequences andinformation regarding the sequences set forth herein. Variouscomputational methods can be implemented to determine the haplotype of asequene. Disclosed herein are computational methods for determiningwhether a sequence is H1, H2, H3, H4, H5, or H6. Current sequencingtechnology can determine haplotypes with tedious and costly experiments.Such restriction makes in silico haplotyping attractive. Many inferenceand statistical methods have been proposed for haplotyping, such asClark method (Clark, A. G. (1990) Inference of haplotypes fromPCR-amplified samples of diploid populations. Molecular Biology andEvolution, 7, 111-122.), parsimony approaches (Gusfield, D. (2001)Inference of haplotypes from samples of diploid populations: Complexityand algorithms. Journal of Computational Biology, 8, 305-324; Lancia,G., Bafna, V., Istrail, S., Lippert, R. and R. Schwartz. (2001) SNPsproblems, complexity and algorithms. In Proceedings of Annual EuropeanSymposium on Algorithms (ESA), 2161, Lecture Notes in Computer Science,182-193, Springer.; Wang, R., Wu, L., Li, Z., Zhang, X. (2005) HaplotypeReconstruction from SNP Fragments by Minimum Error Correction.Bioinformatics, 21, 2456-2462.), maximum-likelihood methods (Excoffier,L. and Slatkin, M. (1995) Maximum-likelihood estimation of molecularhaplotype frequencies in a diploid population. Mol. Biol. Evol., 12,921-927; Hawley, M. and Kidd, K. (1995) Haplo: a program using the EMalgorithm to estimate the frequencies of multi-site haplotypes. J.Heredity, 86, 409-411.), phylogeny-based approaches (Gusfield, D. (2002)Haplotyping as perfect phylogeny: Conceptual framework and efficientsolutions. Proceedings of RECOMB 2002: The sixth Annual InternationalConference on Computational Biology, 166-175.; Chung, R. H. andGusfield, D. (2003) δ5 Perfect phylogeny haplotyper: Haplotype inferralusing a tree model. Bioinformatics, 19, 780-781.; Halperin, E. andEskin, E. (2004) Haplotype reconstruction from genotype data usingimperfect phylogeny. Bioinformatics, 20, 1842-1849.), and Bayesianmethods (Stephens, M., Smith, N. J. and Donnelly, P. (2001) A newstatistical method for haplotype reconstruction from population data.American Journal of Human Genetics, 68, 978-989; Niu, T., Quin, Z. S,Xu, X. and Liu, J. S. (2002) Bayesian haplotype inference for multiplelinked single-nucleotide polymorphisms. American Journal of HumanGenetics, 70, 157-169). In particular, the parsimony criterion thatseeks the minimum number of haplotypes to explain a given set ofgenotypes, has been widely investigated due to its intuitive simplicityand biological implication. Recently both Wang et al. (Wang, L. S, andXu, Y. (2003) Haplotype inference by maximum parsimony. Bioinformatics,19, 1773-1780) and Brown et al. (Wang, L. S, and Xu, Y. (2003) Haplotypeinference by maximum parsimony. Bioinformatics, 19, 1773-1780.)developed an exact algorithm to solve the haplotype inference problembased on the parsimony condition, by the branch-and-bound method and byinteger programming method respectively. However, the pure parsimonyhaplotype inference problem is NP-hard (Gusfield, D. (2001) Inference ofhaplotypes from samples of diploid populations: Complexity andalgorithms. Journal of Computational Biology, 8, 305-324).

10. Kits

Disclosed herein are kits that are drawn to reagents that can be used inpracticing the methods disclosed herein. The kits, can include anyreagent or combination of reagent discussed herein or that would beunderstood to be required or beneficial in the practice of the disclosedmethods. For example, the kits could include primers to perform theamplification reactions discussed in certain embodiments of the methods,as well as the buffers and enzymes required to use the primers asintended. For example, disclosed is a kit for determining a subject'sFVIII haplotype, comprising the oligonucleotides described herein.

11. Blood Plasma Products

Human blood plasma is the yellow, protein-rich fluid that suspends thecellular components of whole blood, that is, the red blood cells, whiteblood cells and platelets. Plasma enables many housekeeping and otherspecialized bodily functions. In blood plasma, the most prevalentprotein is albumin, approximately 32 to 35 grams per liter, which helpsto maintain osmotic balance of the blood.

Blood plasma is generally accumulated in two ways: plasma separated fromdonor collected whole blood, and from donated plasma, a process wherewhole blood is drawn from a donor, the plasma is separated(Plasmapheresis) and then the remainder, less the plasma, is returned tothe donor. The human body completely replaces the lost plasma in amatter of days. FVIII products that are from plasma obtained throughPlasmapheresis are said to be derived from source plasma. FVIII productsthat are from plasma obtained originally from whole blood units (viawhole blood donation), are said to be derived from salvage or recoveredplasma.

Blood plasma, once separated from the other components of whole blood,can be further separated into a number of blood plasma products. Theprocess by which plasma is separated into some of its differentcomponent parts is known as fractionation. Plasma-derived products aremanufactured from batches of blood plasma collected from many thousandsof blood donors. The processing of one pooled lot of plasma can take upto six months and, because of concerns about infectious agents, by rule,the process begins with a 90-day quarantine period. Unlike cellularblood components, blood products derived from plasma can be treated withchemicals, heat, ultraviolet radiation or filtration to decrease costand to increase ease of handling and distribution, and to increase thesafety of the blood supply. Each of these methods has some drawbacks:they may leave unsafe levels of some viruses, be very costly, and/ordamage the blood or blood plasma.

Quantified and defined by volume, albumin continues to be the mainproduct of the blood plasma fractionation industry. Though there aremany uses, its principal use is in restoring blood volume in a widevariety of critical care settings.

Rather than using whole blood transfusions, individual blood componentssuch as red cells, white cells, platelets, and plasma are increasinglybeing used. Plasma is fractionated into an increasing number of bloodplasma products, including albumin, gamma globulins, blood-typing sera,clotting factors (such as FVIU) for people with hemophilia, and more.

Immunoglobins constitute an important class of blood plasma products.This is a group of antibodies made by the body as part of its immuneresponse “team.” These products generally confer immediate, thoughtemporary, protection either from a specific agent, such as rabies virusor snake venom, or arising from a non-specific threat such as in caseswhere an individual's immune system is weakened due to serious illnessor medical treatment which may have an adverse effect on the patient.

Two main groups of immunoglobins are produced intravenous Immunoglobin(IVIG)—A highly heterogeneous product that can provide generalizedimmunity by relying on the inherent variation among individuals and thevariety of immune-provoking agents to which they have been exposed overtheir lifetimes. WIG is made from plasma collected and pooled fromthousands of donors. hyperimmunes—Specific immunoglobins isolated andpurified from selected donors who have strong immunity to a selectedagent. For example, individuals who have been exposed to rabies vaccinecan develop high levels of antibodies to the virus. Immunoglobinsprepared from such plasma can be used as a first treatment when a personhas been bitten by a suspected rabid animal.

The best known of the blood plasma products are the blood clottingfactors, necessary for the wellbeing of those with hemophilia. These canbe derived from donated blood plasma and administered to individuals whoare genetically unable to produce all of the components necessary forblood clotting. The most commonly known need is for Factor VIII.

This pooling of donated blood plasma is made necessary by a newtechnique that substantially reduces costs and potentially facilitatesan easier purifying of the plasma product. The “detergent cleansing”process is not cost effective in small batches.

The New York Blood Center developed this solvent-detergent technology,cleansers that dissolve the fatty coating of viruses such as some HIV,hepatitis B and hepatitis C, some of the so-called lipid-envelopedviruses. It then washes them out of the treated batch.

This process has been used in drugs made from blood plasma, such asimmune globulin or hemophiliacs' clotting factor.

Plasma pooling facilitates the treatment, for purposes of economies ofscale, handling, distribution and blood safety, of collected bloodplasma. This collected and aggregated blood plasma is placed in a commonvat for this process. The process, produces what is known as SolventDetergent Blood Plasma (SD plasma, PLAS+SD). SD blood plasma is a bloodproduct that has undergone treatment with the solvent tri-N-butylphosphate (TNBP) and the detergent Triton X-100 to destroy any lipidbound viruses including: HIV1 and 2, HCV, HBV and HTLVI and II. Theprocess does not destroy non-enveloped viruses such as parvovirus,hepatitis A virus, or any of the prion particles. The SD processincludes the pooling of up to 500,000 units of thawed Fresh Frozen BloodPlasma (FFP), treating it with the solvent and detergent. The treatedblood plasma pool is then sterile filtered (and thus leukocyte-reduced)before being repackaged into 200 mL aliquots or bags and re-frozen. Thisseparation into smaller units is to facilitate handling, distributionand use by the transfusion recipient or the blood product reprocessor.SD Blood plasma can be stored for up to one year frozen at −18° C. Whenordered for transfusion it is thawed in a water bath to a usetemperature of 37° C., which takes approximately 25 to 30 minutes andcan be kept refrigerated for up to 24 hours at 1° to 6° centigrade. OnlyABO identical or compatible SD Blood plasma can be transfused.

Factor VILE concentrates are a commercially prepared, lyophilized powderpurified from human plasma to treat patients with hemostatic disorderssuch as, hemophilia A or von Willebrand's disease. Alternatively,recombinant (synthetic) protein is purified from genetically engineerednon-human cells grown in tissue culture. One factor VIII concentrateunit equals the clotting activity in 1 ml of fresh plasma. Factor VIIIconcentrate is cell free and is administered without regard to patientor donor ABO or Rh type. It is heat treated and/or solvent detergenttreated to reduce the risk of virus transmission. Current processes haveeliminated the risk of HIV, HBV and HCV transmission. Concentratesdiffer in the purification procedures. Highly purified factor VIII,e.g., preparations purified over a monoclonal antibody column or currentrecombinant factor VIII concentrates, are stabilized by adding 98% ofpasteurized human albumin. Porcine factor WI concentrate is availablefor patients with high titer anti-human factor VIII % Bo' orautoantibody inhibitors. Factor VIII concentrates are storedrefrigerated at 35° to 45° Fahrenheit for up to two years from the dateof manufacture. Some preparations may be kept at room temperature forextended periods. Factor VIII concentrate should be infused within fourhours of preparation to reduce the risk of bacterial growth. Vials areusually shipped to a hospital pharmacy, Blood service or nursing unitand mixed there prior to use. Many patients or families receive themdirectly for home care. Methods for purifying FVIII complex from animpure protein fraction are disclosed in U.S. Pat. No. 5,659,017,incorporated herein by reference.

Factor VIII concentrate is indicated for the treatment of bleeding orimminent invasive procedures in patients with hemophilia A, (congenitalfactor VIII deficiency) and for patients with low titer factor VIIIinhibitors. Regular prophylactic doses are often used, as well as dailydoses in some hemophilic inhibitor patients to try to induce immunetolerance. Patients with von Willebrand's disease respond to onespecific, pasteurized intermediate purity concentrate in which thatfactor activity is relatively preserved.

Dosage is dependent on the nature of the injury, the degree of factordeficiency, the weight of the patient and the presence and level orabsence of factor VIII inhibitors. The half life of circulating factorVIII is eight to twelve hours, therefore transfusions may need to berepeated every 12 to 24 hours to maintain hemostatic levels. Followingsurgery, it is necessary to maintain hemostatic levels for up to twoweeks to prevent delayed bleeding and promote wound healing in thehemophilic patient.

Herein described are any combination and permuation of haplotypestogether such as H1 with any combination of H2-H3-H4-H5-H6, H2 with anycombination of H1-H3-H4-H5-H6, H3 with any combination ofH1-H2-H4-H5-H6, H4 with any combination of H1-H2-H3-H5-H6, H5 with anycombination of H1-H2-H3-H5-H6, and H6 with any combination ofH1-H2-H3-H4-H5. For example, combinations can include, but are notlimited to, H1-H2, H1-H2-H3, H1-H2-H3-H4, H1-H2-H3-H4-H5,H1-H2-H3-H4-H5-H6, H2-H3, H2-H3-H4, H2-H3-H4-H5, H2-H3-H4-H5-H6, H3-H4,H3-H4-H5, H3-H4-H5-H6, H4-H5, H4-H5-H6, H5-H6, H1-H3, H1-H4, H1-H5,H2-H4, H2-H5, H2-H6, H3-H5, H3-H6, H4-H6, and the like.

This applies to individuals/subjects that may be heterozygous and haveany combination of haplotypes or wherein pooled blood products cancomprise any of the described combinations and permutations.

D. Methods of Making the Compositions

The compositions disclosed herein and the compositions necessary toperform the disclosed methods can be made using any method known tothose of skill in the art for that particular reagent or compound unlessotherwise specifically noted.

Recombinant factor VIII can be produced through the use of eukaryoticprotein expression systems. In general, a eukaryotic cell line, which isdeficient in a required gene, is transformed with a vector comprisingthe gene that it has a deficiency for, and the recombinant DNA which onewishes to express. Transformation can be accomplished by techniques suchas electroporation or viral delivery. The cell line chosen to producethe protein is selected to be compatible with the protein of interest,capable of continuously expressing the protein of interests, capable ofgrowing on a medium which facilitates purification of the protein ofinterest, along with other factors known to those skilled in the art.Examples of such techniques are disclosed in European Patent Application0 302 968 A2 and U.S. Pat. No. 5,149,637 both of which are incorporatedby reference in their entirety.

The recombinant factor VIII molecules can be tested in humans for theirreduced antigenicity and/or immunogenicity in at least two types ofclinical trials. In one type of trial, designed to determine whether therecombinant or recombinant hybrid factor VIII is immunoreactive withinhibitory antibodies, recombinant or recombinant hybrid factor VIII isadministered, preferably by intravenous infusion, to approximately 25patients having factor VIII deficiency who have antibodies to factorVIII that inhibit the coagulant activity of therapeutic human or porcinefactor VIII. The dosage of the recombinant or recombinant hybrid factorVIII is in a range between 5 and 50 Units/kg body weight, preferably10-50 Units/kg, and most preferably 40 Units/kg body weight.Approximately 1 hour after each administration, the recovery of factorVIII from blood samples is measured in a one-stage coagulation assay.Samples are taken again approximately 5 hours after infusion, andrecovery is measured. Total recovery and the rate of disappearance offactor VIII from the samples is predictive of the antibody titer andinhibitory activity. If the antibody titer is high, factor VIII recoveryusually cannot be measured. The recovery results are compared to therecovery of recovery results in patients treated with plasma-derivedhuman factor VIII, recombinant human factor VIII, porcine factor VIII,and other commonly used therapeutic forms of factor VIII or factor VIIIsubstitutes.

In a second type of clinical trial, designed to determine whether therecombinant or recombinant hybrid factor VIII is immunogenic, i.e.,whether patients will develop inhibitory antibodies, recombinant orrecombinant hybrid factor VIII is administered, as described in thepreceding paragraph, to approximately 100 previously untreatedhemophiliac patients who have not developed antibodies to factor VIII.Treatments are given approximately every 2 weeks over a period of 6months to 1 year. At 1 to 3 month intervals during this period, bloodsamples are drawn and Bethesda assays or other antibody assays areperformed to determine the presence of inhibitory antibodies. Recoveryassays can also be done, after each infusion. Results are compared tohemophiliac patients who receive plasma-derived human factor VIII,recombinant human factor VIII, porcine factor VIII, or other commonlyused therapeutic forms of factor VIII or factor VIII substitutes.

1. Nucleic Acid Synthesis

For example, the nucleic acids, such as, the oligonucleotides to be usedas primers can be made using standard chemical synthesis methods or canbe produced using enzymatic methods or any other known method. Suchmethods can range from standard enzymatic digestion followed bynucleotide fragment isolation (see for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2nd Edition (Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989) Chapters 5, 6) topurely synthetic methods, for example, by the cyanoethyl phosphoramiditemethod using a Milligen or Beckman System 1Plus DNA synthesizer (forexample, Model 8700 automated synthesizer of Milligen-Biosearch,Burlington, Mass. or ABI Model 380B). Synthetic methods useful formaking oligonucleotides are also described by Ikuta et al., Ann. Rev.Biochem. 53:323-356 (1984), (phosphotriester and phosphite-triestermethods), and Narang et al., Methods Enzymol., 65:610-620 (1980),(phosphotriester method). Protein nucleic acid molecules can be madeusing known methods such as those described by Nielsen et al.,Bioconjug. Chem. 5:3-7 (1994).

2. Peptide Synthesis

One method of producing the disclosed proteins, such as SEQ ID NO: 19,is to link two or more peptides or polypeptides together by proteinchemistry techniques. For example, peptides or polypeptides can bechemically synthesized using currently available laboratory equipmentusing either Fmoc (9-fluorenylmethyloxycarbonyl) or Boc(tert-butyloxycarbonoyl) chemistry. (Applied Biosystems, Inc., FosterCity, Calif.). One skilled in the art can readily appreciate that apeptide or polypeptide corresponding to the disclosed proteins, forexample, can be synthesized by standard chemical reactions. For example,a peptide or polypeptide can be synthesized and not cleaved from itssynthesis resin whereas the other fragment of a peptide or protein canbe synthesized and subsequently cleaved from the resin, thereby exposinga terminal group which is functionally blocked on the other fragment. Bypeptide condensation reactions, these two fragments can be covalentlyjoined via a peptide bond at their carboxyl and amino termini,respectively, to form an antibody, or fragment thereof. (Grant G A(1992) Synthetic Peptides: A User Guide. W.H. Freeman and Co., N.Y.(1992); Bodansky M and Trost B., Ed. (1993) Principles of PeptideSynthesis. Springer-Verlag Inc., NY (which is herein incorporated byreference at least for material related to peptide synthesis).Alternatively, the peptide or polypeptide is independently synthesizedin vivo as described herein. Once isolated, these independent peptidesor polypeptides may be linked to form a peptide or fragment thereof viasimilar peptide condensation reactions.

For example, enzymatic ligation of cloned or synthetic peptide segmentsallow relatively short peptide fragments to be joined to produce largerpeptide fragments, polypeptides or whole protein domains (Abrahmsen L etal., Biochemistry, 30:4151 (1991)). Alternatively, native chemicalligation of synthetic peptides can be utilized to syntheticallyconstruct large peptides or polypeptides from shorter peptide fragments.This method consists of a two step chemical reaction (Dawson et al.Synthesis of Proteins by Native Chemical Ligation. Science, 266:776-779(1994)). The first step is the chemoselective reaction of an unprotectedsynthetic peptide—thioester with another unprotected peptide segmentcontaining an amino-terminal Cys residue to give a thioester-linkedintermediate as the initial covalent product. Without a change in thereaction conditions, this intermediate undergoes spontaneous, rapidintramolecular reaction to form a native peptide bond at the ligationsite (Baggiolini M et al. (1992) FEBS Lett. 307:97-101; Clark-Lewis I etal., J. Biol. Chem., 269:16075 (1994); Clark-Lewis I et al.,Biochemistry, 30:3128 (1991); Rajarathnam K et al., Biochemistry33:6623-30 (1994)).

Alternatively, unprotected peptide segments are chemically linked wherethe bond formed between the peptide segments as a result of the chemicalligation is an unnatural (non-peptide) bond (Schnolzer, M et al.Science, 256:221 (1992)). This technique has been used to synthesizeanalogs of protein domains as well as large amounts of relatively pureproteins with full biological activity (deLisle Milton R C et al.,Techniques in Protein Chemistry IV. Academic Press, New York, pp.257-267 (1992)).

3. Process or Making the Compositions

Disclosed are processes for making the compositions as well as makingthe intermediates leading to the compositions There are a variety ofmethods that can be used for making these compositions, such assynthetic chemical methods and standard molecular biology methods. It isunderstood that the methods of making these and the other disclosedcompositions are specifically disclosed.

Disclosed are nucleic acid molecules produced by the process comprisinglinking in an operative way a nucleic acid comprising the sequences setforth herein and a sequence controlling the expression of the nucleicacid.

Also disclosed are nucleic acid molecules produced by the processcomprising linking in an operative way a nucleic acid moleculecomprising a sequence having 80% identity to a sequence set forth inherein, and a sequence controlling the expression of the nucleic acid.

Disclosed are nucleic acid molecules produced by the process comprisinglinking in an operative way a nucleic acid molecule comprising asequence that hybridizes under stringent hybridization conditions to asequence set forth herein and a sequence controlling the expression ofthe nucleic acid.

Disclosed are nucleic acid molecules produced by the process comprisinglinking in an operative way a nucleic acid molecule comprising asequence encoding a peptide set forth herein and a sequence controllingan expression of the nucleic acid molecule.

Disclosed are cells produced by the process of transforming the cellwith any of the disclosed nucleic acids. Disclosed are cells produced bythe process of transforming the cell with any of the non-naturallyoccurring disclosed nucleic acids.

Disclosed are any of the disclosed peptides produced by the process ofexpressing any of the disclosed nucleic acids. Disclosed are any of thenon-naturally occurring disclosed peptides produced by the process ofexpressing any of the disclosed nucleic acids. Disclosed are any of thedisclosed peptides produced by the process of expressing any of thenon-naturally disclosed nucleic acids.

Disclosed are animals produced by the process of transfecting a cellwithin the animal with any of the nucleic acid molecules disclosedherein. Disclosed are animals produced by the process of transfecting acell within the animal any of the nucleic acid molecules disclosedherein, wherein the animal is a mammal. Also disclosed are animalsproduced by the process of transfecting a cell within the animal any ofthe nucleic acid molecules disclosed herein, wherein the mammal ismouse, rat, rabbit, cow, sheep, pig, or primate.

Also disclosed are animals produced by the process of adding to theanimal any of the sequences disclosed herein.

E. Methods of Using the Compositions

In one embodiment, provided are common allelic variants (e.g.haplotypes) of the human factor (F)VIII protein as pharmacogeneticdeterminants that modulate the immunogenicity of wildtype (wt) FVIIIreplacement proteins, the risk of FVIII inhibitor development and theclinical efficacy of transfusion- and gene-delivery-based coagulationfactor replacement therapies.

In another embodiment, provided are common allelic variants (e.g.haplotypes) of the human factor (F)VIII protein as pharmacogeneticdeterminants that modulate thrombosis susceptibility. FIG. 6 is aschematic representation of common non-hemophilic F8 nsSNPs indicativeof thrombosis susceptibility.

Disclosed herein are methods capable of determining the FVIII haplotypeof an individual with a hemostasis disorder where the hemostasisdisorder is thrombophilia (both congenital and acquired).

In a specific embodiment, provided is a haplotype specific Bethesdaassay that overcomes the inherent limitations of the common Bethesdaassay (i.e., near the cutoff point for this assay, under the conditionsin which it is presently performed [with pooled normal plasma, which bydefinition would represent mostly Caucasian donors and therefore mostlyhaplotype 1 wildtype form of the human FVIII protein], this assay isknown to have both a poor specificity [yields false-positives] and apoor sensitivity [yields false-negatives]) as it is used presently.Provided are methods for making normal plasmas that have only one eachof the 6 wildtype forms of the human FVIII protein, so as to minimizethe problems associated with false-negatives and false-positives, and torid any uncertainty about what is the precise make-up of the plasmabeing used.

In one example, a haplotype-specific ELISA assay that is very similar inprinciple to the haplotype-specific Bethesda assay, but can detect allantibodies that bind to the different forms of the wildtype FVIIIprotein, (including those that are functionally neutralizing antibodiescalled inhibitors and those antibodies that are non-inhibitory [i.e.,those that do not inhibit FVIII's function in a clotting assay]), isprovided.

Also provided are kits for genotyping a subset of functional FVIIIpolymorphisms that influence either the circulating levels of FVIIIprotein or activity, and thereby could aid in more accurate diagnosticrisk assessments for the subjects at highest risk for developing venousand/or arterial thrombotic disorders, such as stroke and myocardialinfarction. For example, this can apply to the D1241E haplotype, sincefemales who are carrying two copies of the E-allele will have an ˜25%lower FVIII level than those carrying two major copies of the D-alleleand can have an elevated FVIII level.

Also provided are monoclonal antibody-based assays to rapidly “type” thedifferent haplotypic (allelic) forms of the wildtype FVIII proteindirectly in samples of plasma from patient (in comparison to theDNA-based genotyping assays).

Also provided herein are biological materials, diagnostic materials, andtherapeutic materials.

Disclosed is a method of categorizing a haplotype in a FVIII genecomprising, amplifying regions of the FVIII gene, determining ahaplotype of the FVIII gene from DNA sequence within the amplifiedregions, and categorizing the haplotype as being an H1 (SEQ ID NO: 1),H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO: 4), H5 (SEQ ID NO:5), or H6 (SEQ ID NO: 6).

Disclosed is a method of categorizing a haplotype in a FVIII genecomprising, detecting a FVIII protein and categorizing the haplotype ofthe FVIII gene encoding the detected FVIII protein as being an H1 (SEQID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO: 4), H5(SEQ ID NO: 5), or H6 (SEQ ID NO: 6).

Disclosed is a method of reducing the generation of anti-FVIIIantibodies that inhibit or impair FVIII treatment comprising, detectinga haplotype in a FVIII gene in a subject, matching a replacement FVIIItherapy to the detected haplotype, and administering the matchedreplacement FVIII therapy to the subject. The haplotype can be H1 (SEQID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO: 4), H5(SEQ ID NO: 5), or H6 (SEQ ID NO: 6).

Disclosed is a method of preventing the generation of anti-FVIIIantibodies that inhibit or impair FVIII treatment comprising, detectinga haplotype in a FVIII gene in a subject, matching a replacement FVIIItherapy to the detected haplotype, and administering the matchedreplacement FVIII therapy to the subject. The haplotype can be H1 (SEQID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO: 4), H5(SEQ ID NO: 5), or H6 (SEQ ID NO: 6).

Disclosed is a method of maximizing efficacy of transfusion therapy in asubject with a hemostatic disorder comprising, determining whether theFVIII haplotype of a subject having a hemostatic disorder is H1 (SEQ IDNO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO: 4), H5 (SEQID NO: 5), or H6 (SEQ ID NO: 6) and administering an appropriatetransfusion product to the subject based on the results. The transfusionproduct can be a recombinant FVIII. The transfusion product can beplasma derived FVIII.

Disclosed is a method of administering a blood product to a subject inneed of FVIII comprising, obtaining a haplotype in a FVIII gene of ablood product recipient, determining which type of blood product therecipient should receive based on the result, and administering to thesubject in need thereof an appropriate blood product. The blood productcan be pooled blood plasma derived from more than one blood donor. Theblood product can be a plasma-derived FVIII preparation. The pooledblood plasma can be obtained by detecting a haplotype in a FVIII gene ofa blood plasma donor and placing the blood plasma of the blood plasmadonor in an appropriate FVIII haplotype pool based on the results.

Disclosed is a method of blood plasma pooling comprising, detecting ahaplotype in a FVIII gene of a blood plasma donor and placing bloodplasma of the blood plasma donor in an appropriate pool based on theresults. Disclosed is a pooled blood plasma product obtained throughthis method.

Disclosed is a method of blood plasma pooling comprising, detecting ahaplotype in a FVIII gene of a whole blood donor, receiving whole bloodfrom the whole blood donor, separating plasma from the whole blood, andpooling the plasma with plasma obtained from other donors with a similarhaplotype. Disclosed is a pooled blood plasma product obtained throughthis method.

Disclosed is a method of preparing a plasma-derived FVIII productcomprising, determining the haplotype of blood plasma, wherein thehaplotype is H1 (SEQ ID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4(SEQ ID NO: 4), H5 (SEQ ID NO: 5), or H6 (SEQ ID NO: 6). and preparing aplasma-derived FVIII product from the haplotyped blood plasma whereinthe resulting FVIII product is homogenous with respect to FVIII content.Disclosed is a plasma-derived FVIII product obtained through thismethod.

Disclosed is a method of treating a subject with a hemostatic disordercomprising, identifying a subject with a hemostatic disorder,determining the FVIII haplotype of the subject, wherein the haplotype isH1 (SEQ ID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO:4), H5 (SEQ ID NO: 5), or H6 (SEQ ID NO: 6) and administering anappropriate FVIII gene replacement product to the subject based on theresults. The hemostatic disorder can be congenital hemophilia A. Thehemostatic disorder can be acquired hemophilia A.

The replacement product given will vary, depending on the condition ofthe subject. For example, after determining the FVIII haplotype(s) in apatient with acquired hemophilia A, the disclosed Haplotype-SpecificBethesda assay can be used to determine the haplotype(s) against whichtheir antoanti-FVIII antibodies are reactive. If a given patient'sinhibitors are directed against only their own endogenous FVIIImolecule(s), or react with one or more of the other forms of thewildtype FVIII protein less then with their own, this Bethesda assay canbe used to provide them with the most appropriate replacement FVIIIproduct (i.e., the FVIII haplotype least reactive with their serum).

In congenital hemophilia, antibody inhibitors against these proteins areknown complications of factor replacement therapy. Therefore, thepreviously untreated subject can be given replacement products based ontheir congenital haplotype, thereby maximizing transfusion efficacy inthe treatment of these other hemostatic disorders.

As used herein, the term “appropriate” with regard to a transfusion,blood, or gene replacement product refers to an effective, or useful,haplotype, as well as the amount. Those of skill in the art can readilydetermine an appropriate haplotype based on the methods disclosedherein. Specific dosages and methods of administration are discussedherein as well.

Disclosed is a method of treating a subject with a hemostatic disordercomprising, identifying a subject with a hemostatic disorder,determining the FVIII haplotype of the subject, wherein the haplotype isH1 (SEQ ID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO:4), H5 (SEQ ID NO: 5), or H6 (SEQ ID NO: 6) and administering anappropriate plasma-derived FVIII product to the subject based on theresults. The hemostatic disorder can be congenital hemophilia A. Thehemostatic disorder can be acquired hemophilia A.

Disclosed is a method of treating a subject with a hemostatic disordercomprising, identifying a subject with a hemostatic disorder,determining the FVIII haplotype of the subject, wherein the haplotype isH1 (SEQ ID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO:4), H5 (SEQ ID NO: 5), or H6 (SEQ ID NO: 6) and administering anappropriate recombinant FVIII product to the subject based on theresults. The hemostatic disorder can be congenital hemophilia A. Thehemostatic disorder can be acquired hemophilia A.

Disclosed is a method for rapidly diagnosing a FVIII haplotype in asubject, comprising, obtaining a sample from the subject, analyzing thesample using rapid PCR, and determining a FVIII haplotype for thesubject. The FVIII haplotype can be selected from the group consistingof H1 (SEQ ID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ IDNO: 4), H5 (SEQ ID NO: 5), or H6 (SEQ ID NO: 6). The subject can bediagnosed with congenital hemophilia A. The subject can be diagnosedwith acquired hemophilia A.

Disclosed is a method of maximizing the sensitivity and specificity ofclinical diagnostic algorithms for identifying a subject with aprothrombotic hemostatic disorder comprising, obtaining a sample fromthe subject, determining whether the FVIII haplotype of a subject havinga hemostatic disorder is H1 (SEQ ID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQID NO: 3), H4 (SEQ ID NO: 4), H5 (SEQ ID NO: 5), or H6 (SEQ ID NO: 6)and performing the appropriate additional laboratory diagnostic testingon the subject based on the results.

Disclosed is a method of treating a subject with a prothrombotichemostatic disorder comprising, identifying a subject with a hemostaticdisorder, determining the FVIII haplotype of the subject, wherein thehaplotype is H1 (SEQ ID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4(SEQ ID NO: 4), H5 (SEQ ID NO: 5), or H6 (SEQ ID NO: 6) andadministering an appropriate anti-thrombotic prophylactic treatmentregimen to the subject based on the results. The hemostatic disorder canbe congenital thrombophilia. The hemostatic disorder can be acquiredthrombophilia.

Disclosed are antibodies that target high risk haplotypes of Disclosedare antibodies that target peptide regions unique to said high riskhaplotypes. Disclosed are agents that neutralize the activity of highrisk haplotypes. An agent can be, for example, a monoclonal antibody.Disclosed is an antibody to a polypeptide comprising the sequence as setforth in SEQ ID NO: 19. Disclosed is an n antibody to a polypeptidecomprising the sequence as set forth in SEQ ID NO: 20. Disclosed is an nantibody to a polypeptide comprising the sequence as set forth in SEQ IDNO: 21. Disclosed is an n antibody to a polypeptide comprising thesequence as set forth in SEQ ID NO: 22. Disclosed is an antibody to apolypeptide comprising the sequence as set forth in SEQ ID NO: 23.Disclosed is an antibody to a polypeptide comprising the sequence as setforth in SEQ ID NO: 24.

Disclosed is a method of treating a subject with a prothrombotichemostatic disorder comprising, identifying a subject with aprothrombotic hemostatic disorder, determining the FVIII haplotype ofthe subject, wherein the haplotype is H1 (SEQ ID NO: 1), H2 (SEQ ID NO:2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO: 4), H5 (SEQ ID NO: 5), or H6 (SEQID NO: 6) and administering an appropriate anti-thrombotic prophylactictreatment regimen to the subject based on the results. The hemostaticdisorder can be congenital hemophilia A. The hemostatic disorder can beacquired hemophilia A.

Disclosed is a method of treating a subject with a hemostatic disordercomprising, identifying a subject with a hemostatic disorder,determining the FVIII haplotype of the subject, wherein the haplotype isH1 (SEQ ID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO:4), H5 (SEQ ID NO: 5), or H6 (SEQ ID NO: 6), and administering anappropriate recombinant FVIII product to the subject based on theresults. The hemostatic disorder can be congenital hemophilia A. Thehemostatic disorder can be acquired hemophilia A.

Disclosed is a method for rapidly diagnosing a FVIII haplotype in asubject, comprising, obtaining a sample from the subject, analyzing thesample using rapid PCR, determining a FVIII haplotype for the subject.The FVIII haplotype can be selected from the group consisting of H1 (SEQID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO: 3), H4 (SEQ ID NO: 4), H5(SEQ ID NO: 5), or H6 (SEQ ID NO: 6). The subject can be diagnosed withcongenital hemophilia A. The subject can be diagnosed with acquiredhemophilia A.

Disclosed is a method for rapidly diagnosing a FVIII haplotype in asubject, comprising, obtaining a sample from the subject, analyzing thesample using FVIII haplotype specific antibodies, and determining aFVIII haplotype for the subject. The haplotype can be selected from thegroup consisting of H1 (SEQ ID NO: 1), H2 (SEQ ID NO: 2), H3 (SEQ ID NO:3), H4 (SEQ ID NO: 4), H5 (SEQ ID NO: 5), or H6 (SEQ ID NO: 6). Thesubject can be diagnosed with thrombosis.

Disclosed is a method of screening to determine the presence ofalloantibodies of FVIII comprising, administering FVIII of a knownhaplotype wherein the haplotype is H1 (SEQ ID NO: 1), H2 (SEQ ID NO: 2),H3 (SEQ ID NO: 3), H4 (SEQ ID NO: 4), H5 (SEQ ID NO: 5), H6 (SEQ ID NO:6), and determining whether alloantibody binding occurs.

Disclosed is an oligonucleotide comprising the sequence as set forth inSEQ ID NO: 1. Disclosed is an oligonucleotide comprising the sequence asset forth in SEQ ID NO: 2. Disclosed is an oligonucleotide comprisingthe sequence as set forth in SEQ ID NO: 3. Disclosed is anoligonucleotide comprising the sequence as set forth in SEQ ID NO: 4.Disclosed is an oligonucleotide comprising the sequence as set forth inSEQ ID NO: 5. Disclosed is an oligonucleotide comprising the sequence asset forth in SEQ ID NO: 6.

Disclosed is an oligonucleotide comprising a sequence selected from thegroup consisting of SEQ ID NO:1-12 and 25-124.

Disclosed is a mixture of primers comprising SEQ ID NOS: 25 and 26.Disclosed is a mixture of primers comprising SEQ ID NOS: 29 and 30.Disclosed is a mixture of primers comprising SEQ ID NOS: 33 and 34.Disclosed is a mixture of primers comprising SEQ ID NOS: 37 and 38.Disclosed is a mixture of primers comprising SEQ ID NOS: 25 and 26.Disclosed is a mixture of primers comprising SEQ ID NOS: 41 and 42.Disclosed is a mixture of primers comprising SEQ ID NOS: 43 and 44.Disclosed is a mixture of primers comprising SEQ ID NOS: 45 and 46.Disclosed is a mixture of primers comprising SEQ ID NOS: 47 and 48.Disclosed is a mixture of primers comprising SEQ ID NOS: 49-124.

Disclosed is a polypeptide comprising the sequence as set forth in SEQID NO: 13. Disclosed is a polypeptide comprising the sequence as setforth in SEQ ID NO: 14. Disclosed is a polypeptide comprising thesequence as set forth in SEQ ID NO: 15. Disclosed is a polypeptidecomprising the sequence as set forth in SEQ ID NO: 16. Disclosed is apolypeptide comprising the sequence as set forth in SEQ ID NO: 17.Disclosed is a polypeptide comprising the sequence as set forth in SEQID NO: 18. Disclosed is a polypeptide comprising the sequence as setforth in SEQ ID NO: 19. Disclosed is a polypeptide comprising thesequence as set forth in SEQ ID NO: 20. Disclosed is a polypeptidecomprising the sequence as set forth in SEQ ID NO: 21. Disclosed is apolypeptide comprising the sequence as set forth in SEQ ID NO: 22.Disclosed is a polypeptide comprising the sequence as set forth in SEQID NO: 23. Disclosed is a polypeptide comprising the sequence as setforth in SEQ ID NO: 24.

Disclosed is a composition comprising pooled FVIII having a singlehaplotype. The haplotype can be H1, H2, H3, H4, H5 or H6.

Disclosed is a recombinant FVIII having the H1 haplotype. Disclosed is arecombinant FVIII having the H2 haplotype. Disclosed is a recombinantFVIII having the H3 haplotype. Disclosed is a recombinant FVIII havingthe H4 haplotype. Disclosed is a recombinant FVIR having the H5haplotype. Disclosed is a recombinant FVIII having the H6 haplotype.

Methods of manufacturing recombinant FVIII constructs are disclosed inU.S. Pat. Nos. 6,852,537; 6,770,744; 6,759,216; 6,517,830; 6,458,563;6,376,463; 6,180,371; 5,888,974; 5,859,204; 5,744,446; 5,663,060;5,583,209; 5,364,771 incorporated herein by reference.

1. Methods of Using the Compositions as Research Tools

The disclosed compositions can be used in a variety of ways as researchtools. For example, the disclosed compositions can be used to study thevarious haplotypes of FVIII.

The compositions can be used for example as targets in combinatorialchemistry protocols or other screening protocols to isolate moleculesthat possess desired functional properties related to FVIII.

The disclosed compositions can also be used diagnostic tools related todiseases such as hemophilia and hemostatic disorders, for example.

The disclosed compositions can be used as discussed herein as eitherreagents in micro arrays or as reagents to probe or analyze existingmicroarrays. The disclosed compositions can be used in any known methodfor isolating or identifying single nucleotide polymorphisms. Thecompositions can also be used in any method for determining allelicanalysis of for example, FVIII haplotypes as described herein,particularly allelic analysis as it relates to H1-H6 and theirfunctions. The compositions can also be used in any known method ofscreening assays, related to chip/micro arrays. The compositions canalso be used in any known way of using the computer readable embodimentsof the disclosed compositions, for example, to study relatedness or toperform molecular modeling analysis related to the disclosedcompositions.

2. Methods of Protein Detection

An alternative to genetic haplotyping is haplotyping a subject based offthe protein expressed by their FVIII gene by detecting an FVIII proteinassociated with a disclosed haplotype. The disclosed proteins, includingFVIII proteins in or on any sample, composition or other context, can bedetected using any suitable technique. It is important to be able toseparate FVIII from other proteins, as well as to separate FVIIIsubtypes from each other. Further, molecules that interact with or bindto the disclosed proteins, such as antibodies to a protein, can bedetected using known techniques. Many suitable techniques—such astechniques generally known for the detection of proteins, peptides andother analytes and antigens—are known, some of which are describedbelow. In general, these techniques can involve direct imaging (e.g.,microscopy), immunoassays, or by functional determination. By“functional determination” is meant that a given protein such as aprotein that has a function can be detected by the detection of saidfunction. For example, an enzyme can be detected by evaluating itsactivity on its substrate. The techniques described below are useful indetecting FVIII in a subject, including the various haplotypes describedherein, separating FVIII from other proteins, or in separating varioushaplotypes of FVBI from each other.

a) Immunoassays

Immunodetection methods can be used for detecting, binding, purifying,removing and quantifying various molecules including the disclosed FVIIIhaplotypes. Further, antibodies and ligands to the disclosed proteinscan be detected. For example, the disclosed proteins can be employed todetect antibodies having reactivity therewith. This is useful, forexample, to detect whether a subject has been exposed to or hasdeveloped antibodies against a protein. Standard immunologicaltechniques are described, e.g., in Hertzenberg, et al., Weir's Handbookof Experimental Immunology, vols. 1-4 (1996); Coligan, Current Protocolsin Immunology (1991); Methods in Enzymology, vols. 70, 73, 74, 84, 92,93, 108, 116, 121, 132, 150, 162, and 163; and Paul, FundamentalImmunology (3d ed. 1993) each incorporated herein by reference in itsentirety and specifically for its teaching regarding immunodetectionmethods.

The steps of various useful immunodetection methods have been describedin the scientific literature, such as, e.g., Maggio et al.,Enzyme-Immunoassay, (1987) and Nakamura, et al., Enzyme Immunoassays:Heterogeneous and Homogeneous Systems, Handbook of ExperimentalImmunology, Vol. 1: Immunochemistry, 27.1-27.20 (1986) each incorporatedherein by reference in its entirety and specifically for its teachingregarding immunodetection methods. Immunoassays, in their most simpleand direct sense, are binding assays involving binding betweenantibodies and antigen. Many types and formats of immunoassays are knownand all are suitable for detecting the disclosed proteins. Examples ofimmunoassays are enzyme linked immunosorbent assays (ELISAs),radioimmunoassays (RIA), radioimmune precipitation assays (RIPA),immunobead capture assays, Western blotting, dot blotting, gel-shiftassays, Flow cytometry, protein arrays, multiplexed bead arrays,magnetic capture, in vivo imaging, fluorescence resonance energytransfer (FRET), and fluorescence recovery/localization afterphotobleaching (FRAP/FLAP).

In general, immunoassays involve contacting a sample suspected ofcontaining a molecule of interest (such as the disclosed proteins) withan antibody to the molecule of interest or contacting an antibody to amolecule of interest (such as antibodies to the disclosed proteins) witha molecule that can be bound by the antibody, as the case may be, underconditions effective to allow the formation of immunocomplexes.Contacting a sample with the antibody to the molecule of interest orwith the molecule that can be bound by an antibody to the molecule ofinterest under conditions effective and for a period of time sufficientto allow the formation of immune complexes (primary immune complexes) isgenerally a matter of simply bringing into contact the molecule orantibody and the sample and incubating the mixture for a period of timelong enough for the antibodies to form immune complexes with, i.e., tobind to, any molecules (e.g. antigens) present to which the antibodiescan bind. In many forms of immunoassay, the sample-antibody composition,such as a tissue section, ELISA plate, dot blot or Western blot, canthen be washed to remove any non-specifically bound antibody species,allowing only those antibodies specifically bound within the primaryimmune complexes to be detected.

The sample used can be any sample that is suspected of containing amolecule of interest (or an antibody to a molecule of interest). Thesample can be, for example, one or more cells, tissue, or bodily fluidssuch as blood, urine, semen, lymphatic fluid, cerebrospinal fluid, oramniotic fluid, or other biological samples, such as tissue culturecells, buccal swabs, mouthwash, stool, tissue slices, tissue sections,homogenized tissue extract, cell membrane preparation, biopsyaspiration, archeological samples such as bone or mummified tissue,infection samples, nosocomial infection samples, production samples,drug preparation samples, biological molecule production samples,protein preparation samples, lipid preparation samples, and/orcarbohydrate preparation samples, and separated or purified forms of anyof the above.

Immunoassays can include methods for detecting or quantifying the amountof a molecule of interest (such as the disclosed proteins or theirantibodies) in a sample, which methods generally involve the detectionor quantitation of any immune complexes formed during the bindingprocess. In general, the detection of immunocomplex formation is wellknown in the art and can be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any radioactive, fluorescent, biological orenzymatic tags or any other known label. See, for example, U.S. Pat.Nos. 3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149and 4,366,241, each incorporated herein by reference in its entirety andspecifically for its teaching regarding immunodetection methods andlabels.

As used herein, a label can include a fluorescent dye, a member ofbinding pair, such as biotin/streptavidin, a metal (e.g., gold), or anepitope tag that can specifically interact with a molecule that can bedetected, such as by producing a colored substrate or fluorescence.Substances suitable for detectably labeling proteins include fluorescentdyes (also known herein as fluorochromes and fluorophores) and enzymesthat react with colorometric substrates (e.g., horseradish peroxidase).The use of fluorescent dyes is generally preferred in the practice ofthe invention as they can be detected at very low amounts. Furthermore,in the case where multiple antigens are reacted with a single array,each antigen can be labeled with a distinct fluorescent compound forsimultaneous detection. Labeled spots on the array are detected using afluorimeter, the presence of a signal indicating an antigen bound to aspecific antibody.

Fluorophores are compounds or molecules that luminesce. Typicallyfluorophores absorb electromagnetic energy at one wavelength and emitelectromagnetic energy at a second wavelength. Representativefluorophores include, but are not limited to, 1,5 IAEDANS; 1,8-ANS;4-Methylumbelliferone; 5-carboxy-2,7-dichlorofluorescein;5-Carboxyfluorescein (5-FAM); 5-Carboxynapthofluorescein;5-Carboxytetramethylrhodamine (5-TAMRA); 5-Hydroxy Tryptamine (5-HAT);5-ROX (carboxy-X-rhodamine); 6-Carboxyrhodamine 6G; 6-CR 6G; 6-JOE;7-Amino-4-methylcoumarin; 7-Aminoactinomycin D (7-AAD); 7-Hydroxy-4-Imethylcoumarin; 9-Amino-6-chloro-2-methoxyacridine (ACMA); ABQ; AcidFuchsin; Acridine Orange; Acridine Red; Acridine Yellow; Acriflavin;Acriflavin Feulgen SITSA; Aequorin (Photoprotein); AFPs—AutoFluorescentProtein—(Quantum Biotechnologies) see sgGFP, sgBFP; Alexa Fluor 350™;Alexa Fluor 430™; Alexa Fluor 488™; Alexa Fluor 532™; Alexa Fluor 546™;Alexa Fluor 568™; Alexa Fluor 594™; Alexa Fluor 633™; Alexa Fluor 647™;Alexa Fluor 660™; Alexa Fluor 680™; Alizarin Complexon; Alizarin Red;Allophycocyanin (APC); AMC, AMCA-S; Aminomethylcoumarin (AMCA); AMCA-X;Aminoactinomycin D; Aminocoumarin; Anilin Blue; Anthrocyl stearate;APC-Cy7; APTRA-BTC; APTS; Astrazon Brilliant Red 4G; Astrazon Orange R;Astrazon Red 6B; Astrazon Yellow 7 GLL; Atabrine; ATTO-TAGT™ CBQCA;ATTO-TAG™ FQ; Auramine; Aurophosphine G; Aurophosphine; BAO 9(Bisaminophenyloxadiazole); BCECF (high pH); BCECF (low pH); BerberineSulphate; Beta Lactamase; BFP blue shifted GFP (Y66H); Blue FluorescentProtein; BFP/GFP FRET; Bimane; Bisbenzemide; Bisbenzimide (Hoechst);bis-BTC; Blancophor FFG; Blancophor SV; BOBO™-1; BOBO™⁻³; Bodipy492/515;Bodipy493/503; Bodipy500/510; Bodipy; 505/515; Bodipy 530/550; Bodipy542/563; Bodipy 558/568; Bodipy 564/570; Bodipy 576/589; Bodipy 581/591;Bodipy 630/650-X; Bodipy 650/665-X; Bodipy 665/676; Bodipy F1; Bodipy FLATP; Bodipy F1-Ceramide; Bodipy R6G SE; Bodipy TMR; Bodipy TMR-Xconjugate; Bodipy TMR-X, SE; Bodipy TR; Bodipy TR ATP; Bodipy TR-X SE;BO-PRO™-1; BO-PRO™-3; Brilliant Sulphoflavin FF; BTC; BTC-5N; Calcein;Calcein Blue; Calcium Crimson-; Calcium Green; Calcium Green-1 Ca²⁺ Dye;Calcium Green-2 Ca²⁺; Calcium Green-5N Ca²⁺; Calcium Green-C18 Ca²⁺;Calcium Orange; Calcofluor White; Carboxy-X-rhodamine (5-ROX); CascadeBlue™; Cascade Yellow; Catecholamine; CCF2 (GeneBlazer); CFDA; CFP (CyanFluorescent Protein); CFP/YFP FRET; Chlorophyll; Chromomycin A;Chromomycin A; CL-NERF; CMFDA; Coelenterazine; Coelenterazine cp;Coelenterazine f; Coelenterazine fcp; Coelenterazine h; Coelenterazinehcp; Coelenterazine ip; Coelenterazine n; Coelenterazine O; CoumarinPhalloidin; C-phycocyanine; CPM I Methylcoumarin; CTC; CTC Formazan;Cy2™; Cy3.1 8; Cy3.5™; Cy3™; Cy5.1 8; Cy5.5™; Cy5™; Cy7™; Cyan GFP;cyclic AMP Fluorosensor (FiCRhR); Dabcyl; Dansyl; Dansyl Amine; DansylCadaverine; Dansyl Chloride; Dansyl DHPE; Dansyl fluoride; DAPI;Dapoxyl; Dapoxyl 2; Dapoxyl 3′DCEDA; DCFH (DichlorodihydrofluoresceinDiacetate); DDAO; DHR (Dihydrorhodamine 123); Di-4-ANEPPS; Di-8-ANEPPS(non-ratio); DiA (4-Di 16-ASP); Dichlorodihydrofluorescein Diacetate(DCFH); DiD-Lipophilic Tracer; DiD (DilC18(5)); DIDS; Dihydrorhodamine123 (DHR); Dil (DilC18(3)); I Dinitrophenol; DiO (DiOC18(3)); DiR; DiR(DilC18(7)); DM-NERF (high pH); DNP; Dopamine; DsRed; DTAF; DY-630-NHS;DY-635-NHS; EBFP; ECFP; EGFP; ELF 97; Eosin; Erythrosin; Erythrosin ITC;Ethidium Bromide; Ethidium homodimer-1 (EthD-1); Euchrysin; EukoLight;Europium (111) chloride; EYFP; Fast Blue; FDA; Feulgen (Pararosaniline);FIF (Formaldehyd Induced Fluorescence); FITC; Flazo Orange; Fluo-3;Fluo-4; Fluorescein (Fri C); Fluorescein Diacetate; Fluoro-Emerald;Fluoro-Gold (Hydroxystilbamidine); Fluor-Ruby; Fluor X; FM 1-43™; FM4-46; Fura Red™ (high pH); Fura Red™/Fluo-3; Fura-2; Fura-2/F3CECF;Genacryl Brilliant Red B; Genacryl Brilliant Yellow 10GF; Genacryl Pink3G; Genacryl Yellow 5GF; GeneBlazer; (CCF2); GFP (S65T); GFP red shifted(rsGFP); GFP wild type′ non-UV excitation (wtGFP); GFP wild type, UVexcitation (wtGFP); GFPuv; Gloxalic Acid; Granular blue;Haematoporphyrin; Hoechst 33258; Hoechst 33342; Hoechst 34580; HPTS;Hydroxycoumarin; Hydroxystilbamidine (FluoroGold); Hydroxytryptamine;Indo-1, high calcium; Indo-1 low calcium; Indodicarbocyanine (DiD);Indotricarbocyanine (DiR); Intrawhite Cf; JC-1; JO JO-1; JO-PRO-1;LaserPro; Laurodan; LDS 751 (DNA); LDS 751 (RNA); Leucophor PAF;Leucophor SF; Leucophor WS; Lissamine Rhodamine; Lissamine Rhodamine B;Calcein/Ethidium homodimer; LOLO-1; LO-PRO-1; Lucifer Yellow; LysoTracker Blue; Lyso Tracker Blue-White; Lyso Tracker Green; Lyso TrackerRed; Lyso Tracker Yellow; LysoSensor Blue; LysoSensor Green; LysoSensorYellow/Blue; Mag Green; Magdala Red (Phloxin B); Mag-Fura Red;Mag-Fura-2; Mag-Fura-5; Mag-Indo-1; Magnesium Green; Magnesium Orange;Malachite Green; Marina Blue; I Maxilon Brilliant Flavin 10 GFF; MaxilonBrilliant Flavin 8 GFF; Merocyanin; Methoxycoumarin; Mitotracker GreenFM; Mitotracker Orange; Mitotracker Red; Mitramycin; Monobromobimane;Monobromobimane (mBBr-GSH); Monochlorobimane; MPS (Methyl Green PyronineStilbene); NBD; NBD Amine; Nile Red; Nitrobenzoxedidole; Noradrenaline;Nuclear Fast Red; i Nuclear Yellow; Nylosan Brilliant lavin E8G; OregonGreen™; Oregon Green™ 488; Oregon Green™500; Oregon Green™514; PacificBlue; Pararosaniline (Feulgen); PBFI; PE-Cy5; PE-Cy7; PerCP;PerCP-Cy5.5; PE-TexasRed (Red 613); Phloxin B (Magdala Red); PhorwiteAR; Phorwite BKL; Phorwite Rev; Phorwite RPA; Phosphine 3R; PhotoResist;Phycoerythrin B [PE]; Phycoerythrin R [PE]; PKH26 (Sigma); PKH67; PMIA;Pontochrome Blue Black; POPO-1; POPO-3; PO-PRO-1; PO-I PRO-3; Primuline;Procion Yellow; Propidium lodid (P1); PyMPO; Pyrene; Pyronine; PyronineB; Pyrozal Brilliant Flavin 7GF; QSY 7; Quinacrine Mustard; Resorufin;RH 414; Rhod-2; Rhodamine; Rhodamine 110; Rhodamine 123; Rhodamine 5GLD; Rhodamine 6G; Rhodamine B; Rhodamine B 200; Rhodamine B extra;Rhodamine BB; Rhodamine BG; Rhodamine Green; Rhodamine Phallicidine;Rhodamine: Phalloidine; Rhodamine Red; Rhodamine WT; Rose Bengal;R-phycocyanine; R-phycoerythrin (PE); rsGFP; S65A; S65C; S65L; S65T;Sapphire GFP; SBFI; Serotonin; Sevron Brilliant Red 2B; Sevron BrilliantRed 4G; Sevron I Brilliant Red B; Sevron Orange; Sevron Yellow L; SgBFP™(super glow BFP); sgGFPT™ (super glow GFP); SITS (Primulin; StilbeneIsothiosulphonic Acid); SNAFL calcein; SNAFL-1; SNAFL-2; SNARF calcein;SNARF1; Sodium Green; SpectrumAqua; SpectrumGreen; SpectrumOrange;Spectrum Red; SPQ (6-methoxy-N-(3 sulfopropyl) quinolinium); Stilbene;Sulphorhodamine B and C; Sulphorhodamine Extra; SYTO 11; SYTO 12; SYTO13; SYTO 14; SYTO 15; SYTO 16; SYTO 17; SYTO 18; SYTO 20; SYTO 21; SYTO22; SYTO 23; SYTO 24; SYTO 25; SYTO 40; SYTO 41; SYTO 42; SYTO 43; SYTO44; SYTO 45; SYTO 59; SYTO 60; SYTO 61; SYTO 62; SYTO 63; SYTO 64; SYTO80; SYTO 81; SYTO 82; SYTO 83; SYTO 84; SYTO 85; SYTOX Blue; SYTOXGreen; SYTOX Orange; Tetracycline; Tetramethylrhodamine (TRITC); TexasRed™; Texas Red-X™ conjugate; Thiadicarbocyanine (DiSC3); Thiazine RedR; Thiazole Orange; Thioflavin 5; Thioflavin S; Thioflavin TON;Thiolyte; Thiozole Orange; Tinopol CBS (Calcofluor White); TIER;TO-PRO-1; TO-PRO-3; TO-PRO-5; TOTO-1; TOTO-3; TriColor (PE-Cy5); TRITCTetramethylRodaminelsoThioCyanate; True Blue; Tru Red; Ultralite;Uranine B; Uvitex SFC; wt GFP; WW 781; X-Rhodamine; XRITC; XyleneOrange; Y66F; Y66H; Y66W; Yellow GFP; YFP; YO-PRO-1; YO-PRO3; YOYO-1;YOYO-3; Sybr Green; Thiazole orange (interchelating dyes); semiconductornanoparticles such as quantum dots; or caged fluorophore (which can beactivated with light or other electromagnetic energy source), or acombination thereof.

Green flourescent protein (GFP) has become the one of the most popularreporter proteins and as such a nice epitope tag. However, GFP is muchlarger than most other epitope tags.

Digoxigenin (DIG) is a small organic molecule that can be covalentlyadded to proteins or nucleic acids, which makes it useful in diverseapplications.

Biotin is a small molecule that can be covalently linked to proteinsafter they have been translated. Therefore, unlike most other proteinepitope tags, it can be added at any point in time and is often used tolabel proteins located in particular sites such as on the extracellularsurface of cells.

Flourescent dyes, such as those described herein, for which antibodiesare available are also commonly used as epitope tags.

Labeling can be either direct or indirect. In direct labeling, thedetecting antibody (the antibody for the molecule of interest) ordetecting molecule (the molecule that can be bound by an antibody to themolecule of interest) include a label. Detection of the label indicatesthe presence of the detecting antibody or detecting molecule which inturn indicates the presence of the molecule of interest or of anantibody to the molecule of interest, respectively. In indirectlabeling, an additional molecule or moiety is brought into contact with,or generated at the site of, the immunocomplex. For example, asignal-generating molecule or moiety such as an enzyme can be attachedto or associated with the detecting antibody or detecting molecule. Thesignal-generating molecule can then generate a detectable signal at thesite of the immunocomplex. For example, an enzyme, when supplied withsuitable substrate, can produce a visible or detectable product at thesite of the immunocomplex. ELISAs use this type of indirect labeling.

As another example of indirect labeling, an additional molecule (whichcan be referred to as a binding agent) that can bind to either themolecule of interest or to the antibody to the molecule of interest,such as a second antibody to the molecule of interest, can be contactedwith the immunocomplex. The additional molecule can have a label orsignal-generating molecule or moiety. The additional molecule can be anantibody, which can thus be termed a secondary antibody. Binding of asecondary antibody to the molecule of interest can form a so-calledsandwich with the first (or primary) antibody and the molecule ofinterest. The immune complexes can be contacted with the labeled,secondary antibody under conditions effective and for a period of timesufficient to allow the formation of secondary immune complexes. Thesecondary immune complexes can then be generally washed to remove anynon-specifically bound labeled secondary antibodies, and the remaininglabel in the secondary immune complexes can then be detected. Theadditional molecule can also be or include one of a pair of molecules ormoieties that can bind to each other, such as the biotin/avadin pair. Inthis mode, the detecting antibody or detecting molecule should includethe other member of the pair.

Other modes of indirect labeling include the detection of primary immunecomplexes by a two step approach. For example, a molecule (which can bereferred to as a first binding agent), such as an antibody, that hasbinding affinity for the molecule of interest or corresponding antibodycan be used to form secondary immune complexes, as described above.After washing, the secondary immune complexes can be contacted withanother molecule (which can be referred to as a second binding agent)that has binding affinity for the first binding agent, again underconditions effective and for a period of time sufficient to allow theformation of immune complexes (thus forming tertiary immune complexes).The second binding agent can be linked to a detectable label orsignal-generating molecule or moiety, allowing detection of the tertiaryimmune complexes thus formed. This system can provide for signalamplification.

Immunoassays that involve the detection of as substance, such as aprotein or an antibody to a specific protein, include label-free assays,protein separation methods (i.e., electrophoresis), solid supportcapture assays, or in vivo detection. Label-free assays are generallydiagnostic means of determining the presence or absence of a specificprotein, or an antibody to a specific protein, in a sample. Proteinseparation methods are additionally useful for evaluating physicalproperties of the protein, such as size or net charge. Capture assaysare generally more useful for quantitatively evaluating theconcentration of a specific protein, or antibody to a specific protein,in a sample. Finally, in vivo detection is useful for evaluating thespatial expression patterns of the substance, i.e., where the substancecan be found in a subject, tissue or cell.

b) Label-Free Assays

Provided that the concentrations are sufficient, the molecular complexes([Ab—Ag]n) generated by antibody-antigen interaction are visible to thenaked eye, but smaller amounts may also be detected and measured due totheir ability to scatter a beam of light. The formation of complexesindicates that both reactants are present, and in immunoprecipitationassays a constant concentration of a reagent antibody is used to measurespecific antigen ([Ab—Ag]n), and reagent antigens are used to detectspecific antibody ([Ab—Ag]n). If the reagent species is previouslycoated onto cells (as in hemagglutination assay) or very small particles(as in latex agglutination assay), “clumping” of the coated particles isvisible at much lower concentrations. A variety of assays based on theseelementary principles are in common use, including Ouchterlonyimmunodiffusion assay, rocket immunoelectrophoresis, andimmunoturbidometric and nephelometric assays. The main limitations ofsuch assays are restricted sensitivity (lower detection limits) incomparison to assays employing labels and, in some cases, the fact thatvery high concentrations of analyte can actually inhibit complexformation, necessitating safeguards that make the procedures morecomplex. Some of these Group 1 assays date right back to the discoveryof antibodies and none of them have an actual “label” (e.g. Ag-enz).Other kinds of immunoassays that are label free depend on immunosensors,and a variety of instruments that can directly detect antibody-antigeninteractions are now commercially available. Most depend on generatingan evanescent wave on a sensor surface with immobilized ligand, whichallows continuous monitoring of binding to the ligand. Immunosensorsallow the easy investigation of kinetic interactions and, with theadvent of lower-cost specialized instruments, may in the future findwide application in immunoanalysis.

c) Protein Separation

The use of immunoassays to detect a specific protein can involve theseparation of the proteins by electophoresis. This can be done toseparate FVIII from other proteins, as well as separating FVIII subtypesfrom each other. Electrophoresis is the migration of charged moleculesin solution in response to an electric field. Their rate of migrationdepends on the strength of the field; on the net charge, size and shapeof the molecules and also on the ionic strength, viscosity andtemperature of the medium in which the molecules are moving. As ananalytical tool, electrophoresis is simple, rapid and highly sensitive.It is used analytically to study the properties of a single chargedspecies, and as a separation technique.

Generally the sample is run in a support matrix such as paper, celluloseacetate, starch gel, agarose or polyacrylamide gel. The matrix inhibitsconvective mixing caused by heating and provides a record of theelectrophoretic run: at the end of the run, the matrix can be stainedand used for scanning, autoradiography or storage. In addition, the mostcommonly used support matrices—agarose and polyacrylamide—provide ameans of separating molecules by size, in that they are porous gels. Aporous gel may act as a sieve by retarding, or in some cases completelyobstructing, the movement of large macromolecules while allowing smallermolecules to migrate freely. Because dilute agarose gels are generallymore rigid and easy to handle than polyacrylamide of the sameconcentration, agarose is used to separate larger macromolecules such asnucleic acids, large proteins and protein complexes. Polyacrylamide,which is easy to handle and to make at higher concentrations, is used toseparate most proteins and small oligonucleotides that require a smallgel pore size for retardation.

Proteins are amphoteric compounds; their net charge therefore isdetermined by the pH of the medium in which they are suspended. In asolution with a pH above its isoelectric point, a protein has a netnegative charge and migrates towards the anode in an electrical field.Below its isoelectric point, the protein is positively charged andmigrates towards the cathode. The net charge carried by a protein is inaddition independent of its size—i.e., the charge carried per unit mass(or length, given proteins and nucleic acids are linear macromolecules)of molecule differs from protein to protein. At a given pH therefore,and under non-denaturing conditions, the electrophoretic separation ofproteins is determined by both size and charge of the molecules.

Sodium dodecyl sulphate (SDS) is an anionic detergent which denaturesproteins by “wrapping around” the polypeptide backbone—and SDS binds toproteins fairly specifically in a mass ratio of 1.4:1. In so doing, SDSconfers a negative charge to the polypeptide in proportion to itslength. Further, it is usually necessary to reduce disulphide bridges inproteins (denature) before they adopt the random-coil configurationnecessary for separation by size: this is done with 2-mercaptoethanol ordithiothreitol (DTT). In denaturing SDS-PAGE separations therefore,migration is determined not by intrinsic electrical charge of thepolypeptide, but by molecular weight.

Determination of Molecular Weight is done by SDS-PAGE of proteins ofknown molecular weight along with the protein to be characterized. Alinear relationship exists between the logarithm of the molecular weightof an SDS-denatured polypeptide, or native nucleic acid, and its Rf. TheRf is calculated as the ratio of the distance migrated by the moleculeto that migrated by a marker dye-front. A simple way of determiningrelative molecular weight by electrophoresis (Mr) is to plot a standardcurve of distance migrated vs. log 10 MW for known samples, and read offthe log Mr of the sample after measuring distance migrated on the samegel.

In two-dimensional electrophoresis, proteins are fractionated first onthe basis of one physical property, and, in a second step, on the basisof another. For example, isoelectric focusing can be used for the firstdimension, conveniently carried out in a tube gel, and SDSelectrophoresis in a slab gel can be used for the second dimension. Oneexample of a procedure is that of O'Farrell, P. H., High ResolutionTwo-dimensional Electrophoresis of Proteins, J. Biol. Chem.250:4007-4021 (1975), herein incorporated by reference in its entiretyfor its teaching regarding two-dimensional electrophoresis methods.Other examples include but are not limited to, those found in Anderson,L and Anderson, NG, High resolution two-dimensional electrophoresis ofhuman plasma proteins, Proc. Natl. Acad. Sci. 74:5421-5425 (1977),Ornstein, L., Disc electrophoresis, L. Ann. N.Y. Acad. Sci. 121:321349(1964), each herein incorporated by reference in its entirety for itsteaching regarding electrophoresis methods.

Laemmli, U. K., Cleavage of structural proteins during the assembly ofthe head of bacteriophage T4, Nature 227:680 (1970), herein incorporatedby reference in its entirety for its teaching regarding electrophoresismethods, discloses a discontinuous system for resolving proteinsdenatured with SDS. The leading ion in the Laemmli buffer system ischloride, and the trailing ion is glycine. Accordingly, the resolvinggel and the stacking gel are made up in Tris-HCl buffers (of differentconcentration and pH), while the tank buffer is Tris-glycine. Allbuffers contain 0.1% SDS.

Western Blot

One example of an immunoassay that uses electrophoresis that iscontemplated in the current methods is Western Blot analysis. Westernblot can be used to detect FVBJ subtypes, for example. Western blottingor immunoblotting allows the determination of the molecular mass of aprotein and the measurement of relative amounts of the protein presentin different samples. Detection methods include chemiluminescence andchromagenic detection. Standard methods for Western Blot analysis can befound in, for example, D. M. Bollag et al., Protein Methods (2d edition1996) and E. Harlow & D. Lane, Antibodies, a Laboratory Manual (1988),U.S. Pat. No. 4,452,901, each herein incorporated by reference in theirentirety for their teaching regarding Western Blot methods. Generally,proteins are separated by gel electrophoresis, usually SDS-PAGE. Theproteins are transferred to a sheet of special blotting paper, e.g.,nitrocellulose, though other types of paper, or membranes, can be used.The proteins retain the same pattern of separation they had on the gel.The blot is incubated with a generic protein (such as milk proteins) tobind to any remaining sticky places on the nitrocellulose. An antibodyis then added to the solution which is able to bind to its specificprotein.

The attachment of specific antibodies to specific immobilized antigenscan be readily visualized by indirect enzyme immunoassay techniques,usually using a chromogenic substrate (e.g. alkaline phosphatase orhorseradish peroxidase) or chemiluminescent substrates. Otherpossibilities for probing include the use of fluorescent or radioisotopelabels (e.g., fluorescein, ¹²⁵I). Probes for the detection of antibodybinding can be conjugated anti-immunoglobulins, conjugatedstaphylococcal Protein A (binds IgG), or probes to biotinylated primaryantibodies (e.g., conjugated avidin/streptavidin).

The power of the technique lies in the simultaneous detection of aspecific protein by means of its antigenicity, and its molecular mass:proteins are first separated by mass in the SDS-PAGE, then specificallydetected in the immunoassay step. Thus, protein standards (ladders) canbe run simultaneously in order to approximate molecular mass of theprotein of interest in a heterogeneous sample.

d) Radioimmune Precipitation Assay (RIPA)

Radioimmune Precipitation Assay (RIPA) is a sensitive assay usingradiolabeled antigens to detect specific antibodies in serum. Theantigens are allowed to react with the serum and then precipitated usinga special reagent such as, for example, protein A sepharose beads. Thebound radiolabeled immunoprecipitate is then commonly analyzed by gelelectrophoresis. Radioimmunoprecipitation assay (RIPA) is often used asa confirmatory test for diagnosing the presence of HIV antibodies. RIPAis also referred to in the art as Farr Assay, Precipitin Assay,Radioimmune Precipitin Assay; Radioimmunoprecipitation Analysis;Radioimmunoprecipitation Analysis, and RadioimmunoprecipitationAnalysis.

e) Capture Assays

While the above immunoassays that utilize electrophoresis to separateand detect the specific proteins of interest allow for evaluation ofprotein size, they are not very sensitive for evaluating proteinconcentration. However, also contemplated are immunoassays wherein theprotein or antibody specific for the protein is bound to a solid support(e.g., tube, well, bead, or cell) to capture the antibody or protein ofinterest, respectively, from a sample, combined with a method ofdetecting the protein or antibody specific for the protein on thesupport. Examples of such immunoassays include Radioimmunoassay (RIA),Enzyme-Linked Immunosorbent Assay (ELISA), Flow cytometry, proteinarray, multiplexed bead assay, and magnetic capture.

(1) Radioimmunoassay (RIA)

Radioimmunoassay (RIA) is a classic quantitative assay for detection ofantigen-antibody reactions using a radioactively labeled substance(radioligand), either directly or indirectly, to measure the binding ofthe unlabeled substance to a specific antibody or other receptor system.Radioimmunoassay is used, for example, to test hormone levels in theblood without the need to use a bioassay. Non-immunogenic substances(e.g., haptens) can also be measured if coupled to larger carrierproteins (e.g., bovine gamma-globulin or human serum albumin) capable ofinducing antibody formation.

RIA involves mixing a radioactive antigen (because of the ease withwhich iodine atoms can be introduced into tyrosine residues in aprotein, the radioactive isotopes ¹²⁵I or ¹³¹I are often used) withantibody to that antigen. The antibody is generally linked to a solidsupport, such as the tube or beads. Unlabeled or “cold” antigen is thenadding in known quantities and measuring the amount of labeled antigendisplaced. Initially, the radioactive antigen is bound to theantibodies. When cold antigen is added, the two compete for antibodybinding sites—and at higher concentrations of cold antigen, more bindsto the antibody, displacing the radioactive variant. The bound antigensare separated from the unbound ones in solution and the radioactivity ofeach used to plot a binding curve. The technique is both extremelysensitive, and specific.

(2) ELISAs

Enzyme-Linked Immunosorbent Assay (ELISA), or more generically termedEIA (Enzyme ImmunoAssay), is an immunoassay that can detect an antibodyspecific for a protein. ELISA is useful with the methods disclosedherein, in that the FVIII protein can be detected using this assay,including but not limited to specific haplotypes of FVIII. In such anassay, a detectable label bound to either an antibody-binding orantigen-binding reagent is an enzyme. When exposed to its substrate,this enzyme reacts in such a manner as to produce a chemical moietywhich can be detected, for example, by spectrophotometric, fluorometricor visual means. Enzymes which can be used to detectably label reagentsuseful for detection include, but are not limited to, horseradishperoxidase, alkaline phosphatase, glucose oxidase, β-galactosidase,ribonuclease, urease, catalase, malate dehydrogenase, staphylococcalnuclease, asparaginase, yeast alcohol dehydrogenase,alpha.-glycerophosphate dehydrogenase, triose phosphate isomerase,glucose-6-phosphate dehydrogenase, glucoamylase andacetylcholinesterase. For descriptions of ELISA procedures, see Voller,A. et al., J. Clin. Pathol. 31:507-520 (1978); Butler, J. E., Meth.Enzymol. 73:482-523 (1981); Maggio, E. (ed.), Enzyme Immunoassay, CRCPress, Boca Raton, 1980; Butler, J. E., In: Structure of Antigens, Vol.1 (Van Regenmortel, M., CRC Press, Boca Raton, 1992, pp. 209-259;Butler, J. E., In: van Oss, C. J. et al., (eds), Immunochemistry, MarcelDekker, Inc., New York, 1994, pp. 759-803; Butler, J. E. (ed.),Immunochemistry of Solid-Phase Immunoassay, CRC Press, Boca Raton,1991); Crowther, “ELISA: Theory and Practice,” In: Methods in MoleculeBiology, Vol. 42, Humana Press; New Jersey, 1995; U.S. Pat. No.4,376,110, each incorporated herein by reference in its entirety andspecifically for its teaching regarding ELISA methods.

Variations of ELISA techniques are know to those of skill in the art. Inone variation, antibodies that can bind to proteins can be immobilizedonto a selected surface exhibiting protein affinity, such as a well in apolystyrene microtiter plate. Then, a test composition suspected ofcontaining a marker antigen can be added to the wells. After binding andwashing to remove non-specifically bound immunocomplexes, the boundantigen can be detected. Detection can be achieved by the addition of asecond antibody specific for the target protein, which is linked to adetectable label. This type of ELISA is a simple “sandwich ELISA”.Detection also can be achieved by the addition of a second antibody,followed by the addition of a third antibody that has binding affinityfor the second antibody, with the third antibody being linked to adetectable label.

Another variation is a competition ELISA. In competition ELISA's, testsamples compete for binding with known amounts of labeled antigens orantibodies. The amount of reactive species in the sample can bedetermined by mixing the sample with the known labeled species before orduring incubation with coated wells. The presence of reactive species inthe sample acts to reduce the amount of labeled species available forbinding to the well and thus reduces the ultimate signal.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating or binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes.Antigen or antibodies can be linked to a solid support, such as in theform of plate, beads, dipstick, membrane or column matrix, and thesample to be analyzed applied to the immobilized antigen or antibody. Incoating a plate with either antigen or antibody, one will generallyincubate the wells of the plate with a solution of the antigen orantibody, either overnight or for a specified period of hours. The wellsof the plate can then be washed to remove incompletely adsorbedmaterial. Any remaining available surfaces of the wells can then be“coated” with a nonspecific protein that is antigenically neutral withregard to the test antisera. These include bovine serum albumin (BSA),casein and solutions of milk powder. The coating allows for blocking ofnonspecific adsorption sites on the immobilizing surface and thusreduces the background caused by nonspecific binding of antisera ontothe surface.

In BLISAs, a secondary or tertiary detection means rather than a directprocedure can also be used. Thus, after binding of a protein or antibodyto the well, coating with a non-reactive material to reduce background,and washing to remove unbound material, the immobilizing surface iscontacted with the control clinical or biological sample to be testedunder conditions effective to allow immunecomplex (antigen/antibody)formation. Detection of the immunecomplex then requires a labeledsecondary binding agent, or a secondary binding agent in conjunctionwith a labeled third binding agent.

“Under conditions effective to allow immunecomplex (antigen/antibody)formation” means that the conditions include diluting the antigens andantibodies with solutions such as BSA, bovine gamma globulin (BGG) andphosphate buffered saline (PBS)/Tween. These added agents can alsoassist in the reduction of nonspecific background.

The “suitable” conditions also mean that the incubation is at atemperature and for a period of time sufficient to allow effectivebinding. Incubation steps can typically be from about 1 minute to twelvehours, at temperatures of about 20° to 30° C., or can be incubatedovernight at about 0° C. to about 10° C.

Following all incubation steps in an ELISA, the contacted surface can bewashed so as to remove non-complexed material. A washing procedure caninclude washing with a solution such as PBS/Tween, or borate buffer.Following the formation of specific immunecomplexes between the testsample and the originally bound material, and subsequent washing, theoccurrence of even minute amounts of immunecomplexes can be determined.

To provide a detecting means, the second or third antibody can have anassociated label to allow detection, as described above. This can be anenzyme that can generate color development upon incubating with anappropriate chromogenic substrate. Thus, for example, one can contactand incubate the first or second immunecomplex with a labeled antibodyfor a period of time and under conditions that favor the development offurther immunecomplex formation (e.g., incubation for 2 hours at roomtemperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label can be quantified, e.g., byincubation with a chromogenic substrate such as urea and bromocresolpurple or 2,2′-azido-di-(3-ethyl-benzthiazoline-6-sulfonic acid [ABTS]and H₂O₂, in the case of peroxidase as the enzyme label. Quantitationcan then be achieved by measuring the degree of color generation, e.g.,using a visible spectra spectrophotometer.

(3) Flow Cytometry

Flow cytometry can be used to detect various FVIII haplotypes, and canbe used to purify mixed samples of haplotypes, as disclosed herein. Flowcytometry, fluorescent activated cell sorting (FACS), or flowmicrofluorometry provides the means of scanning individual cells for thepresence of a molecule of interest. Flow Cytometry is thecharacterization of single cells as they pass at high speed through alaser beam. While a hematologist can count 200 cells in less than aminute by hand (hemocytometer) on a stage microscope, a flow cytometercan discriminate cells at speeds up to 50,000 cells/second. The Flowcomponent is a fluidics system that precisely delivers the cells at theintersection of the laser beam and light gathering lens by hydrodynamicfocusing (a single stream of cells is injected and confined within anouter stream at greater pressure). The laser acting as a light sourcedevelops parameters of light scatter as well as exciting the fluorescentmolecules used to label the cell. Cells are characterized individuallyby their physical and/or chemical properties (Kohler, G. and Milstein,C. (1975) Continuous Cultures of Fused Cells Secreting Antibody ofPredefined Specificity. Nature 256: p. 495-49) which provide analyticalparameters capable of accurate quantitation of the number ofmolecules/cell through Quantitative Flow Cytometry (QFCM). The physical(morphological) profile of a cell can be observed by combining forwardlight scatter (FS) and orthogonal or side light scatter (SSC). Inforward light scatter the laser beam is interrupted by the cell and thelight that passes around the cell is measured. Comparable to castingshadow puppets on a wall with a flashlight. This measurement is anindication of the cell's unique refractive index which depends on acell's size, organelles, water and molecular contents. The refractiveindex (forward scatter) of a cell can change through cell cycleprogression, activation, fixation, etc. Cellular side scatter is thelight that is reflected 90° to the laser beam (all fluorescence isemitted and therefore collected at this angle) and is an indication ofcytoplasmic density or cell surface granularity.

A short list of some of the information that can be discerned bymultiparameter (multi-color) Flow Cytometry includes; Apoptosis(programmed cell death), Cell Type, DNA Content, Enzyme Activity,Intracellular Proteins, Cell Surface Antigens, Cytoplasmic Granularity,Surface Membrane Integrity, Intracellular [Ca++]-Signal Transduction,DNA Synthesis-Proliferation, Cell Surface Receptors, IntracellularCytokines, Oxidative Metabolism, Intracellular pH, RNA Content, and CellSize.

Antibodies can provide a useful tool for the analysis and quantitationof markers of individual cells. Such flow cytometric analyses aredescribed in Melamed, et al., Flow Cytometry and Sorting (1990);Shapiro, Practical Flow Cytometry (1988); and Robinson, et al., Handbookof Flow Cytometry Methods (1993), each herein incorporated by referencein its entirety for their teaching regarding FACS. Generally, proteinsare detected with antibodies that have been conjugated to fluorescentmolecules such'as FITC, PE, Texas Red, APC, etc. Detection of moleculeson the cell surface (immunophenotyping) is most common, but with a fewmodifications, proteins can be identified in the cytoplasm (e.g.cytokines), or in the nucleus (e.g. cell cycle control proteins).

By tagging antibodies with a colored fluorochrome, it is easy todistinguish the cell type and quantity of antigens expressed by eachcell. Employing dichroic splitting mirrors, band pass filters andcompensation, the colors can be resolved where each color is associatedwith a single antibody. As each cell, tagged with a fluorescentlylabeled antibody, enters the laser light outer orbital electrons in thefluorochrome are excited at a specific excitation wavelength (e.g., 494nm for FITC). As it transitions the width of the laser beam maximum peakfluorescence is achieved within approximately 10 nsec as the excitedouter orbital electrons return to their more stable ground state andemit a photon of light at a longer wavelength (e.g., 520 nm for FITC)than that at which they were excited. Photomultiplier tubes (PMT's)detect these faint fluorescent signals and their sole role is to changediscrete packets of light called photons QM into electrons and amplifythem by producing as much as 10 million electrons for every photoncaptured.

In addition, cytoplasmic and nuclear membranes can be breached(permeabilized) to introduce, for example, antibodies againstintracellular proteins or stain nucleic acids with intercalating dyes(propidium iodide, 7-AAD, etc.) or DNA base specific dyes (Hoescht). Inaddition, BrdU incorporation for cell proliferation can be measured byimmunofluorescence. DNA intercalating dyes, such as for examplepropidium iodide, allow the identification of which cells are diploid(G0, resting, non-dividing cells, or any cycling cells in G1), whichcells are in S phase of the cell cycle during which DNA is replicated,and which cells are G2 or in mitosis. Tumor cells are oftenaneuploid—that is, they have too many or too few chromosomes compared tothe normal diploid DNA number for the species.

Apoptosis can be measured in a number of ways. The TUNEL techniqueidentifies DNA strand breaks. Annexin V immunolabeling detects changesin the plasma membrane asymmetry. Nuclear condensation and DNA loss aredemonstrated by hypodiploid peaks in DNA content experiments. Uptake ofHoechst 33258 has also been shown to increase with apoptotic changes.

Fluorescence-activated cell sorting (FACS) is a type of flow cytometry.FACS is a method for sorting a suspension of biologic cells into two ormore containers, one cell at a time. Fluorescence-activated cell sortingis based upon specific light scattering and fluorescence characteristicsof each cell. In FACS, the cell suspension is entrained in the center ofa narrow, rapidly flowing stream of liquid. The flow is arranged so thatthere is a large separation between cells relative to their diameter. Avibrating mechanism causes the stream of cells to break into individualdroplets. The system is adjusted so that there is a low probability ofmore than one cell being in a droplet. Just before the stream breaksinto droplets the flow passes through a fluorescence measuring stationwhere the fluorescence character of interest of each cell is measured.An electrical charging ring is placed just at the point where the streambreaks into droplets. A charge is placed on the ring based on theimmediately prior fluorescence intensity measurement and the oppositecharge is trapped on the droplet as it breaks from the stream. Thecharged droplets then fall through an electrostatic deflection systemthat diverts droplets into containers based upon their charge.

(4) Protein Arrays

Protein arrays are useful with the methods disclosed herein as assaysfor detection of various FVIII haplotypes in an individual or incollective samples, for example. Protein arrays are solid-phase ligandbinding assay systems using immobilised proteins on surfaces whichinclude glass, membranes, microliter wells, mass spectrometer plates,and beads or other particles. The assays are highly parallel(multiplexed) and often miniaturised (microarrays, protein chips). Theiradvantages include being rapid and automatable, capable of highsensitivity, economical on reagents, and giving an abundance of data fora single experiment. Bioinformatics support is important; the datahandling demands sophisticated software and data comparison analysis.However, the software can be adapted from that used for DNA arrays, ascan much of the hardware and detection systems.

One of the chief formats is the capture array, in which ligand-bindingreagents, which are usually antibodies but can also be alternativeprotein scaffolds, peptides or nucleic acid aptamers, are used to detecttarget molecules in mixtures such as plasma or tissue extracts. Indiagnostics, capture arrays can be used to carry out multipleimmunoassays in parallel, both testing for several analytes inindividual sera for example and testing many serum samplessimultaneously. In proteomics, capture arrays are used to quantitate andcompare the levels of proteins in different samples in health anddisease, i.e., protein expression profiling. Proteins other thanspecific ligand binders are used in the array format for in vitrofunctional interaction screens such as protein-protein, protein-DNA,protein-drug, receptor-ligand, enzyme-substrate, etc. They may also beused to correlate the polymorphic changes resulting from SNPs withprotein function. The capture reagents themselves are selected andscreened against many proteins, which can also be done in a multiplexarray format against multiple protein targets.

For construction of arrays, sources of proteins include cell-basedexpression systems for recombinant proteins, purification from naturalsources, production in vitro by cell-free translation systems, andsynthetic methods for peptides. Many of these methods can be automatedfor high throughput production. For capture arrays and protein functionanalysis, it is important that proteins should be correctly folded andfunctional; this is not always the case, e.g. where recombinant proteinsare extracted from bacteria under denaturing conditions. Nevertheless,arrays of denatured proteins are useful in screening antibodies forcross-reactivity, identifying autoantibodies and selecting ligandbinding proteins.

Protein arrays have been designed as a miniaturisation of familiarimmunoassay methods such as ELISA and dot blotting, often utilizingfluorescent readout, and facilitated by robotics and high throughputdetection systems to enable multiple assays to be carried out inparallel. Commonly used physical supports include glass slides, silicon,microwells, nitrocellulose or PVDF membranes, and magnetic and othermicrobeads. While microdrops of protein delivered onto planar surfacesare the most familiar format, alternative architectures include CDcentrifugation devices based on developments in microfluidics [Gyros]and specialised chip designs, such as engineered microchannels in aplate [The Living Chip™, Biotrove] and tiny 3D posts on a siliconsurface [Zyomyx]. Particles in suspension can also be used as the basisof arrays, providing they are coded for identification; systems includecolour coding for microbeads [Luminex, Bio-Rad] and semiconductornanocrystals [QDots™, Quantum Dots], and barcoding for beads[UltraPlex™, Smartbeads] and multimetal microrods [Nanobarcodes™particles, Nanoplex Technologies]. Beads can also be assembled intoplanar arrays on semiconductor chips [LEAPS technology, BioArraySolutions].

Immobilization of proteins involves both the coupling reagent and thenature of the surface being coupled to. A good protein array supportsurface is chemically stable before and after the coupling procedures,allows good spot morphology, displays minimal nonspecific binding, doesnot contribute a background in detection systems, and is compatible withdifferent detection systems. The immobilization method used arereproducible, applicable to proteins of different properties (size,hydrophilic, hydrophobic), amenable to high throughput and automation,and compatible with retention of fully functional protein activity.Orientation of the surface-bound protein is recognized as an importantfactor in presenting it to ligand or substrate in an active state; forcapture arrays the most efficient binding results are obtained withorientated capture reagents, which generally require site-specificlabeling of the protein.

Both covalent and noncovalent methods of protein immobilization are usedand have various pros and cons. Passive adsorption to surfaces ismethodologically simple, but allows little quantitative or orientationalcontrol; it may or may not alter the functional properties of theprotein, and reproducibility and efficiency are variable. Covalentcoupling methods provide a stable linkage, can be applied to a range ofproteins and have good reproducibility; however, orientation may bevariable, chemical derivatization may alter the function of the proteinand requires a stable interactive surface. Biological capture methodsutilizing a tag on the protein provide a stable linkage and bind theprotein specifically and in reproducible orientation, but the biologicalreagent must first be immobilized adequately and the array may requirespecial handling and have variable stability.

Several immobilization chemistries and tags have been described forfabrication of protein arrays. Substrates for covalent attachmentinclude glass slides coated with amino- or aldehyde-containing silanereagents. In the Versalinx™ system [Prolinx], reversible covalentcoupling is achieved by interaction between the protein derivatised withphenyldiboronic acid, and salicylhydroxamic acid immobilized on thesupport surface. This also has low background binding and low intrinsicfluorescence and allows the immobilized proteins to retain function.Noncovalent binding of unmodified protein occurs within porousstructures such as HydroGel™ [PerkinEhner], based on a 3-dimensionalpolyacrylamide gel; this substrate is reported to give a particularlylow background on glass microarrays, with a high capacity and retentionof protein function. Widely used biological coupling methods are throughbiotin/streptavidin or hexahistidine/Ni interactions, having modifiedthe protein appropriately. Biotin may be conjugated to a poly-lysinebackbone immobilised on a surface such as titanium dioxide [Zyomyx] ortantalum pentoxide [Zeptosens].

Array fabrication methods include robotic contact printing, ink-jetting,piezoelectric spotting and photolithography. A number of commercialarrayers are available [e.g. Packard Biosience] as well as manualequipment [V & P Scientific]. Bacterial colonies can be roboticallygridded onto PVDF membranes for induction of protein expression in situ.

At the limit of spot size and density are nanoarrays, with spots on thenanometer spatial scale, enabling thousands of reactions to be performedon a single chip less than 1 mm square. BioForce Laboratories havedeveloped nanoarrays with 1521 protein spots in 85 sq microns,equivalent to 25 million spots per sq cm, at the limit for opticaldetection; their readout methods are fluorescence and atomic forcemicroscopy (AFM).

Fluorescence labeling and detection methods are widely used. The sameinstrumentation as used for reading DNA microarrays is applicable toprotein arrays. For differential display, capture (e.g. antibody) arrayscan be probed with fluorescently labeled proteins from two differentcell states, in which cell lysates are directly conjugated withdifferent fluorophores (e.g. Cy-3, Cy-5) and mixed, such that the coloracts as a readout for changes in target abundance. Fluorescent readoutsensitivity can be amplified 10-100 fold by tyramide signalamplification (TSA) [PerkinElmer Lifesciences]. Planar waveguidetechnology [Zeptosens] enables ultrasensitive fluorescence detection,with the additional advantage of no intervening washing procedures. Highsensitivity can also be achieved with suspension beads and particles,using phycoerythrin as label [Luminex] or the properties ofsemiconductor nanocrystals [Quantum Dot]. A number of novel alternativereadouts have been developed, especially in the commercial biotecharena. These include adaptations of surface plasmon resonance [HTSBiosystems, Intrinsic Bioprobes], rolling circle DNA amplification[Molecular Staging], mass spectrometry [Ciphergen, Intrinsic Bioprobes],resonance light scattering [Genicon Sciences] and atomic forcemicroscopy [BioForce Laboratories].

Capture arrays form the basis of diagnostic chips and arrays forexpression profiling. They employ high affinity capture reagents, suchas conventional antibodies, single domains, engineered scaffolds,peptides or nucleic acid aptamers, to bind and detect specific targetligands in high throughput manner.

Antibody arrays have the required properties of specificity andacceptable background, and some are available commercially [BDBiosciences Clontech, BioRad, Sigma]. Antibodies for capture arrays aremade either by conventional immunisation (polyclonal sera andhybridomas), or as recombinant fragments, usually expressed in E. coli,after selection from phage or ribosome display libraries [CambridgeAntibody Technology, BioInvent, Affitech, Biosite]. In addition to theconventional antibodies, Fab and scFv fragments, single V-domains fromcamelids or engineered human equivalents [Domantis] may also be usefulin arrays.

The term ‘scaffold’ refers to ligand-binding domains of proteins, whichare engineered into multiple variants capable of binding diverse targetmolecules with antibody-like properties of specificity and affinity. Thevariants can be produced in a genetic library format and selectedagainst individual targets by phage, bacterial or ribosome display. Suchligand-binding scaffolds or frameworks include ‘Affibodies’ based onStaph. aureus protein A [Affibody], ‘Trinectins’ based on fibronectins[Phylos] and ‘Anticalins’ based on the lipocalin structure [Pieris].These can be used on capture arrays in a similar fashion to antibodiesand may have advantages of robustness and ease of production.

Nonprotein capture molecules, notably the single-stranded nucleic acidaptamers which bind protein ligands with high specificity and affinity,are also used in arrays [SomaLogic]. Aptamers are selected fromlibraries of oligonucleotides by the Selex™ procedure and theirinteraction with protein can be enhanced by covalent attachment, throughincorporation of brominated deoxyuridine and UV-activated crosslinking(photoaptamers). Photocrosslinking to ligand reduces the crossreactivityof aptamers due to the specific steric requirements. Aptamers have theadvantages of ease of production by automated oligonucleotide synthesisand the stability and robustness of DNA; on photoaptamer arrays,universal fluorescent protein stains can be used to detect binding.

Protein analytes binding to antibody arrays may be detected directly orvia a secondary antibody in a sandwich assay. Direct labelling is usedfor comparison of different samples with different colours. Where pairsof antibodies directed at the same protein ligand are available,sandwich immunoassays provide high specificity and sensitivity and aretherefore the method of choice for low abundance proteins such ascytokines; they also give the possibility of detection of proteinmodifications. Label-free detection methods, including massspectrometry, surface plasmon resonance and atomic force microscopy,avoid alteration of ligand. What is required from any method is optimalsensitivity and specificity, with low background to give high signal tonoise. Since analyte concentrations cover a wide range, sensitivity hasto be tailored appropriately; serial dilution of the sample or use ofantibodies of different affinities are solutions to this problem.Proteins of interest are frequently those in low concentration in bodyfluids and extracts, requiring detection in the pg range or lower, suchas cytokines or the low expression products in cells.

The question of cross-reactivity is an important one which applies toall ligand binders and particularly to antibodies, being the mostpopular reagents. While antibodies are thought of as being highlyspecific, monoclonals can show unpredictable cross-reactions which willbe revealed by thorough screening. The ultimate usefulness of individualreagents then depends on the relative level of cross-reaction andspecific reaction. The use of sandwich assays, in which antibody pairsare used to bind and detect ligand, may go a long way towardseliminating the problem, since it is unlikely that both members of thesandwich will exhibit the same cross-reactivity. Polyclonal antibodiesare emerging as array reagents for protein expression studies; althoughthey require affinity purification, rabbit sera are easier to producethan monoclonals, and cross-reactions may be reduced as a result ofheterogeneity. There are ambitious projects to raise monoclonalantibodies and antisera against the entire human proteome.

An important general principle is that, for optimal specificity whereassays are highly multiplexed, it is essential to provide dual leveltarget recognition, i.e. two levels of specificity for each locus in thearray. Sandwich assays achieve this with two antibodies,photocrosslinking reduces the cross-reactivity of aptamers and MSprovides definitive label-free protein identification.

An alternative to an array of capture molecules is one made through‘molecular imprinting’ technology, in which peptides (e.g. from theC-terminal regions of proteins) are used as templates to generatestructurally complementary, sequence-specific cavities in apolymerisable matrix; the cavities can then specifically capture(denatured) proteins which have the appropriate primary amino acidsequence [ProteinPrint™, Aspira Biosystems].

Another methodology which can be used diagnostically and in expressionprofiling is the ProteinChip® array [Ciphergen], in which solid phasechromatographic surfaces bind proteins with similar characteristics ofcharge or hydrophobicity from mixtures such as plasma or tumourextracts, and SELDI-TOF mass spectrometry is used to detection theretained proteins. The ProteinChip® is credited with the ability toidentify novel disease markers. However, this technology differs fromthe protein arrays under discussion here since, in general, it does notinvolve immobilisation of individual proteins for detection of specificligand interactions.

Large-scale functional chips have been constructed by immobilising largenumbers of purified proteins and used to assay a wide range ofbiochemical functions, such as protein interactions with other proteins,drug-target interactions, enzyme-substrates, etc. Generally they requirean expression library, cloned into E. coli, yeast or similar from whichthe expressed proteins are then purified, e.g. via a His tag, andimmobilised. Cell free protein transcription/translation is a viablealternative for synthesis of proteins which do not express well inbacterial or other in vivo systems.

For detecting protein-protein interactions, protein arrays can be invitro alternatives to the cell-based yeast two-hybrid system and may beuseful where the latter is deficient, such as interactions involvingsecreted proteins or proteins with disulphide bridges. High-throughputanalysis of biochemical activities on arrays has been described foryeast protein kinases and for various functions (protein-protein andprotein-lipid interactions) of the yeast proteome, where a largeproportion of all yeast open-reading frames was expressed andimmobilised on a microarray. Large-scale ‘proteome chips’ promise to bevery useful in identification of functional interactions, drugscreening, etc. [Proteometrix]. Another possible screen will be for theeffect of polymorphisms arising from disease-related coding SNPs (SAPs,single amino acid polymorphisms); such information may be valuable inascertaining the effects of SNPs on drug responses and side effects inpatients (pharmacogenomics). One restriction is that proteins which areonly functional as multicomponent complexes will probably not beanalysable on protein arrays.

As a two-dimensional display of individual elements, a protein array canbe used to screen phage or ribosome display libraries, in order toselect specific binding partners, including antibodies, syntheticscaffolds, peptides and aptamers. In this way, ‘library against library’screening can be carried out. Screening of drug candidates incombinatorial chemical libraries against an array of protein targetsidentified from genome projects is another application of the approach.

(5) Multiplexed Bead Assay

A multiplexed bead assay, such as for example the BD™ Cytometric BeadArray, is a series of spectrally discrete particles that can be used tocapture and quantitate soluble analytes. The analyte is then measured bydetection of a fluorescence-based emission and flow cytometric analysis.Multiplexed bead assay generates data that is comparable to ELISA basedassays, but in a “multiplexed” or simultaneous fashion. Concentration ofunknowns is calculated for the cytometric bead array as with anysandwich format assay, i.e. through the use of known standards andplotting unknowns against a standard curve. Further, multiplexed beadassay allows quantification of soluble analytes in samples neverpreviously considered due to sample volume limitations. In addition tothe quantitative data, powerful visual images can be generated revealingunique profiles or signatures that provide the user with additionalinformation at a glance.

(6) Magnetic Capture

Antibody-coated magnetic particles can be used to capture andselectively separate target cells or specific chemicals from solution.In the technique, target-specific antibody is bound to a magneticparticle (often termed an immunobead). After reaction time to allowbinding of immunobead and target, a strong magnetic field is applied toselectively separate the captured target-particle complexes from themilieu.

(7) Immunocytochemistry/Immunohistochemistry

Also provided are methods of detecting a substance of interest such as aprotein, such as FVIII subtypes, in vivo or in situ using antibodyconjugates. Immunocytochemistry and immunohistochemistry are techniquesfor identifying cellular or tissue constituents, respectively, by meansof antigen-antibody interactions. The methods generally involveadministering to an animal or subject an imaging-effective amount of adetectably-labeled protein-specific antibody or fragment thereof, andthen detecting the location of the labeled antibody in the sample cellor tissue. An “imaging effective amount” is an amount of adetectably-labeled antibody, or fragment thereof, that when administeredis sufficient to enable later detection of binding of the antibody orfragment in the specific cell or tissue. The effective amount of theantibody-marker conjugate is allowed sufficient time to come intocontact with reactive antigens that are present within the tissues ofthe subject, and the subject is then exposed to a detection device toidentify the detectable marker.

Administration of the antibodies can be done as disclosed herein.Nucleic acid approaches for antibody delivery also exist. The antibodiesand antibody fragments disclosed herein can also be administered topatients or subjects as a nucleic acid preparation (e.g., DNA or RNA)that encodes the antibody or antibody fragment, such that the patient'sor subject's own cells take up the nucleic acid and produce and secretethe encoded antibody or antibody fragment. The delivery of the nucleicacid can be by any means, as disclosed herein, for example.Administration of the antibody can be local or systemic and accomplishedintravenously, intra-arterially, via the spinal fluid or the like.Administration also can be intradermal or intracavitary, depending uponthe body site under examination. After a sufficient time has lapsed forthe labeled antibody or fragment to bind to the diseased tissue, forexample 30 minutes to 48 hours, the area of the subject underinvestigation can then be examined by an imaging technique, such asthose described herein.

The distribution of the bound radioactive isotope and its increase ordecrease with time can be monitored and recorded. By comparing theresults with data obtained from studies of clinically normalindividuals, the presence and extent of the diseased tissue can bedetermined.

The exact imaging protocol will necessarily vary depending upon factorsspecific to the subject, and depending upon the body site underexamination, method of administration, type of label used and the like.One of ordinary skill in the art will be able to determine which imagingprotocol to use based on these factors. Effective dosages and schedulesfor administering the compositions can be determined empirically, andmaking such determinations is within the skill in the art. The dosageranges for the administration of the compositions are those large enoughto produce the desired effect in which the symptoms of the disorder areaffected. The dosage should not be so large as to cause adverse sideeffects, such as unwanted cross-reactions, anaphylactic reactions, andthe like. Generally, the dosage will vary with the age, condition, sexand extent of the disease in the patient, route of administration, orwhether other drugs are included in the regimen, and can be determinedby one of skill in the art. The dosage can be adjusted by the individualphysician in the event of any counterindications. Dosage can vary, andcan be administered in one or more dose administrations daily, for oneor several days. Guidance can be found in the literature for appropriatedosages for given classes of pharmaceutical products. For example,guidance in selecting appropriate doses for antibodies can be found inthe literature on therapeutic uses of antibodies, e.g., Ferrone et al.,Handbook of Monoclonal Antibodies, (1985) ch. 22 and pp. 303-357; Haberet al., Antibodies in Human Diagnosis and Therapy, (1977) pp. 365-389. Atypical daily dosage of the antibody used alone might range from about 1μg/kg to up to 100 mg/kg of body weight or more per day, depending onthe factors mentioned above.

Methods of Factor Inhibitor Detection (Bethesda Assay)

Inhibitor to Factor VIII is screened for by mixing test plasma with aknown amount of Factor VIII. After a 2 hour incubation period at 37° C.,the residual Factor VIII activity is determined in a factor VIII assay.By comparing the difference in the Factor VIII activity of the patientincubation mixture and a control mixture, the absence or presence of aFactor VIII inhibitor can be demonstrated. Some antibodies will onlyprolong the Protein Truncation Test (PTT) after incubation. The assaydisclosed herein can utilize blood plasma that contains only one of the6 wildtype form of FVIII protein.

Factor VIII inhibitors, IgG antibodies directed against factor VIII, canoccur in alloimmunized patients with congenital factor VIII deficiency(Hemophilia A) or as autoantibodies. The latter are associated withpregnancy, autoimmune disease, or drugs but most often occurspontaneously, particularly among elderly persons.

General steps involved in a Bethesda assay for factor VIII inhibitorinclude: Serial subject plasma dilutions in citrated saline areprepared, from 1:1 up to 1:160 (or higher if necessary for high-titerfactor inhibitors). The purpose of these dilutions is to dilute out theinhibitor. The patient plasma dilutions are then mixed with an equalvolume of normal plasma containing a normal amount of coagulationfactors. The mixed dilutions are usually incubated for up to 2 hours,because certain inhibitors show an inhibitory effect only afterprolonged incubation (particularly factor V and factor VIII inhibitors).Factor VIII assays are then performed on each mixed dilution. Thedilution that inhibits 50% of factor VIII in the assay defines the titerof the inhibitor. For example, if the 1:40 dilution inhibits 50% of thefactor VIII in the assay, the patient is reported to have a titer of 40BU of factor VIII inhibitor.

Porcine factor VIII can be substituted for normal plasma (which containshuman factor VIII) in the Bethesda assay to determine if the factor VIIIinhibitor cross-reacts with porcine factor VIII. If there is little orno cross-reactivity, porcine factor VIII is often used to treat bleedingdue to a factor VIII inhibitor.

The Bethesda assay can be modified to identify and titer other specificfactor inhibitors. For example, if a factor V inhibitor is suspected,factor V assays are performed on the mixed dilutions instead of factorVIII assays.

Antibodies that inhibit the activity of a specific coagulation factorcan develop spontaneously or in association with certain medications,autoimmune diseases, or other conditions. These antibodies may alsoarise when a patient with a hereditary factor deficiency is transfusedwith a product containing the factor, such as a factor concentrate orfresh frozen plasma. The immune system in the patient with thedeficiency views the transfused factor as foreign, and forms an antibodyagainst the transfused factor. This complication makes treatment ofbleeding episodes difficult in such patients. The most common clinicallysignificant factor inhibitor is a factor VIII inhibitor. Factor VIIIinhibitors cause decreased factor VIII activity and consequently aprolonged PTT. Factor VIII inhibitors exhibit a characteristic patternin the PTT mixing study where the mixed plasma PTT is initially normal(or significantly more normal than the patient plasma's PTT) but becomesprolonged (typically by increasing at least 8-10 seconds) over thecourse of a 1- to 2-hour incubation.

Use of the Haplotype-Specific Bethesda Assay in Patients with AcquiredHemophilia A

When a non-hemophilic patient presents with bleeding, whether or not itis reminiscent of hemorrhagic episodes seen in hemophilia patients, theyundergo a diagnostic evaluation for identifying the common acquiredbleeding disorders via testing performed in the Clinical HemostasisLaboratory. Inhibitors are usually first detected using a sensitiveclotting-based assay, variably referred to as an inhibitor screen or amixing study. If a coagulation factor inhibitor is suspected from theresults of the screening assay, the specificity of these antibodies(i.e., anti-FVIII-vs anti-FIX-vs anti-FV-vs anti-vWF-antibodies) isusually determined by performing clotting-based assays that are specificfor the different coagulation factors. Once the presence and specificityof the suspected inhibitor has been determined, their presence andspecificity are most often confirmed by performing the more specificclotting-based test known as the Bethesda assay. The plasma level (i.e.titer) of an inhibitor, likewise, is typically measured by the Bethesdaassay and is defined in terms of Bethesda units (BU).

While use of the traditional Bethesda assay, as described above, canconfirm the specificity of an inhibitor as recognizing FVIII, and notanother coagulation factor, because it is based on pooled normal plasmaas the “wildtype FVIII source”, one cannot determine the specificity ofthe inhibitor towards the six structurally-distinct forms of thewildtype FVIII protein expressed by humans. Using the Haplotype-SpecificBethesda assay disclosed herein, the reactivity of the auto-FVIIIantibodies can be specifically evaluated in a patient with acquiredhemophilia A, towards the different forms of the human FVIII protein.These autoantibodies, by definition, should be directed against thepatient's own (i.e. “self”) FVIII molecule(s). Acquired hemophilia A, incontrast to the congenital form, affects both males and females,although it has been observed more frequently in women then men. Sincefemales have two X-chromosomes and therefore two F8, the wildtype FVIIIprotein they express may all represent the same wildtype form (i.e.haplotype) or two distinct haplotypes of the protein. Therefore, theautoanti-FVIII antibodies in female patients with acquired hemophilia Amay represent either one, or at most two, of the six naturally-occurringforms of the human FVIII. After determining the FVIII haplotype(s) in apatient with acquired hemophilia A, the disclosed Haplotype-SpecificBethesda assay can be used to determine the haplotype(s) against whichtheir antoanti-FVIII antibodies are reactive. If a given patient'sinhibitors are directed against only their own endogenous FVIIImolecule(s), or react with one or more of the other forms of thewildtype FVIII protein less then with their own, this Bethesda assay canbe used to provide them with the most appropriate replacement FVIIIproduct (i.e., the FVIII haplotype least reactive with their serum).

a) Detection and Quantification of Inhibitors

Factor replacement therapy, the intravenous infusion of concentrated,purified preparations of either recombinant (r) or plasma-derived (p)wildtype human FVIII, is the most widely used and effective means ofarresting and/or preventing bleeding in patients with hemophilia A.Unfortunately, the exogenous FVBI protein is seen as a foreign in ˜25%of patients and becomes targeted by functionally neutralizingalloantibodies, termed inhibitors. Because inhibitors can leave patientsrefractory to further treatment with FVIII, and necessitate the use ofalternative therapies that are less desirable from a clinical andeconomic standpoint, the development of such antibodies is considered tobe the most serious complication in the management of hemophilia A.Therefore, all patients with hemophilia A should be periodically testedfor the presence of inhibitors.

Different methods have been used to detect and quantify inhibitors, aswell as non-inhibitory anti-FVIII antibodies. Typically, however, atraditional Bethesda assay, with or without the Njiemegan modification,has been performed on all hemophilia A patients testing positive in an aPTT-based plasma mixing study, which are often referred to as aninhibitor screen. Dilutions of a patient's plasma is then mixed with apooled normal plasma (PNP), which is derived from a large collection ofhealthy unrelated blood donors and therefore contains wildtype FVIII,and incubated at 37° C. for two hours. At the end of the two hours, aFVIII activity (FVIII:C) assay is performed on the mixtures, using areagent plasma deficient for FVIII:C, so that the amount of residualFVIII:C can be calculated. This method has also been used to detectother factor inhibitors through the use of PNP that has beenimmunodepleted of the coagulation protein suspected of being the target.

The Bethesda assay has long been known to have several shortcomings,which include, among others, a poor sensitivity to low-titre antibodiesand a poor specificity near the cut-off. While this has been attributed,at least in part, to the complex reaction kinetics of some inhibitors,this needs to be re-investigated in light of the recent discovery thathumans express at least six structurally-distinct forms of the wildtypeFVIII protein. Since the Bethesda assay uses PNP as the source of FVIIIto detect inhibitors in patient plasmas, it is clear that this finding,which demonstrates as inaccurate the long-held view that FVIII ismonomorphic at the protein level in the non-hemophilic human population,could account for at least some of the problems associated with theBethesda assay.

Assay principle: An inhibitor to FVIII, or more accurately isquantitated by mixing the patient's plasma, which is known as the testplasma, with a FVIII source plasma, that has typically been a PNPcontaining a known amount of “wildtype” FVIII:C. After an incubationperiod, the residual FVBI:C activity level is measured in an aPTT-basedspecific FVIII actrivity assay that also requires FVIII deficientplasma. By comparing the difference in FVIII:C levels of the patientincubation mixtures and a control mixture, the amount of inhibitorpresent can be calculated in Bethesda units (BU's). One BU of inhibitoris defined as the amount of inhibitor that will inactivate 50% of theoriginal FVIII:C level present.

The novel Haplotype-Specific Bethesda assay incorporates the recentdiscovery that, due to the presence of four non-synonymous SNPs (nsSNPs)in the FVIII gene (F8), humans express six wildtype forms of thecoagulation FVIII protein, referred to as haplotypes H1, H2, H3, H4, H5,and H6. Through the use of immunodepleted FVIII-deficient human plasmasthat have been reconstituted with purified recombinant (r) FVIIIpreparations that represent one of the six forms of the human protein,different homogeneous FVIII source plasmas are available that can beused to maximize the sensitive of this ssay in a given patient bytesting their plasma for antibodies against only the form of themolecule(s) that they have been treated with. This novel strategysimultaneously increases the specificity of the traditional Bethesdaassay, by also testing a patient's plasma against a source plasmacontaining only the wildtype FVIII protein corresponding to their ownbackground form of the molecule.

b) Specimen Requirements

A. Collect patient's blood in two B-D blue-top siliconized Vacutainertubes containing 0.5 mL of 0.1M buffered sodium citrate (3.2% sodiumcitrate).

B. If drawing with a syringe, use a 2-syringe technique, discardingblood in the first syringe or using it for other tests. Blood for testshould come from the second syringe.

C. If using Vacutainer collection, at least one other tube should becollected before collecting tubes for the test.

D. The collection tube must contain ˜4.5 mL of blood. Theplasma-to-anticoagulant ratio is critical. No short-sampled oroverfilled tubes will be accepted.

E. Sample should be brought to the Hemostasis Lab immediately aftercollection, with time of collection marked on the label. If specimencannot be delivered to the Lab immediately place on ice. If specimen ison ice for >4 hours, it will not be accepted for testing.

F. Specimen should be centrifuged immediately with top on tube at 2,500g for 15 minutes (or adjusted speed/time to provide residual plateletsof <10×10⁹/L).

G. Using plastic dispo pipet, remove platelet-free plasma to plastic12×75 tubes and cover.

H. If the assay cannot be performed within 4 hours, the plasma must befrozen at −20° C. (stable for 2 weeks) or −70° C. (stable for 6 months).Frozen plasma must be thawed rapidly in a 37° C. waterbath when ready toperform test.

I. Severely hemolyzed specimens will not be accepted for testing.

c) Equipment & Reagents

A. Equipment:

Stago-STA or other comparable automated coagulomator

Centrifuge

Pipettes

Pipette tips

Plastic test tubes

Cuvette roll (contains 1000 STA cuvettes)

Magnetic stir bars

Bar-coded Information sheet

Reagent grade waxer

B. Reagents:

1. STA-Neoplastine-CI-Plus® Reagent (extrinsic pathway factorinhibitors)a. Reagent 1: STA-Neoplastin-CI-Plus® thromboplastin prepared from freshrabbit cerebral tissues. The ISI value of this reagent is determinedagainst a secondary standard known as the BCT (British comparativethromboplastin), and is indicated in the bar-code insert provided in thebox.b. Reagent 2: Solvent containing calcium with sodium azide as apreservative, 10 mL per vial.c. Transfer reagent 2 into the vial of reagent 1 of same lot #. Swirlgently to obtain a homogeneous suspension. Keep the reconstitutedreagent in the original vial and allow it to stand at room temperature(18-25° C.) for 30 minutes before use.d. Before placing the reagent on the instrument, remove rubber stopper,add a stir bar to the vial, and install the perforated plastic cap onthe vial. The mixed reagent is stable 48 hours on the STA. Do notfreeze.2. PTT-A (intrinsic pathway factor inhibitors): reconstitute each vialwith 5 mL of distilled water. Allow the reconstituted material to standat room temperature for 30 minutes. Swirl the reagent gently, place amagnet inside, and install the perforated plastic cap on the vial.Vortex bottle before use. Stable for 24 hours on the STA. Do not freeze.3. CaCL2 (0.025M); no reconstitution necessary. Stable for 5 days on theSTA.4. Sterile, filtered, distilled water.5. Imidazole buffered saline: weigh in a 1 L volumetric flask 3.4 gimidazole and 5.85 g sodium chloride; add 186 mL 0.1N hydrochloric acid,until pH is at 7.3 and qs to 1 L with sterile water. The solution isstored at 2-8° C.6. 0.1N HCl; measure 8.3 mL concentrated hydrochloric acid into a 1000mL flask (volumetric) and qs to 1000 mL with sterile water. The solutionis stored at 2-8° C.7. Pooled normal plasma: George King pooled normalplasma-(unassayed)-George King Biomedical. Store frozen at −70° C. Thawrapidly in 37° C. waterbath, vortex and keep cold until use.8. Recombinant haplotype 1 human factor VIII (r-hFVIII-H1): dissolve abottle of lyophilized r-hFVIII-H1 to a final concentration of 20Units/mL with sterile filtered water. Example: if the concentration onthe bottle is 475 Units (may vary)

${X\mspace{14mu} {mL}} = {\frac{475\mspace{14mu} {Units}}{20\mspace{14mu} {Units}\text{/}{mL}} = {23.75\mspace{14mu} {mL}}}$

-   -   X=volume of sterile water to add to bottle to achieve 20        Units/mL Distribute the reconstituted r-hFVIII-H1 via 125 μL        aliquots into labeled cyrovials and freeze at −70° C. Stable        indefinately under this storage condition. Thaw for 5 minutes at        37° C. in a waterbath immediately prior to using.        9. R-hFVIIII-H1 plasma (1 Unit/mL): make this working solution        by adding 50 μL of the stock r-hFVIII-H1 solution (20 Units/mL)        to 950 μL of immunodepleted FVIII deficient human plasma (i.e.,        NoFact-VIIIi; R²—Diagnostics, South Bend, Ind.; see below).        10. Recombinant haplotype 2 human factor VIII (r-hFVIII-H2):        repeat procedure in step 8 above to reconstitute a vial of        lyophylized r-hFVIII-H2 at 20 Units/mL        11. R-hFVIII-H2 plasma (1 Unit/mL): repeat procedure in step 9        above to prepare a working solution of r-hFVIII-H2 (20 Units/mL)        12. Repeat steps 8 and 9 individually four more times to prepare        identical working solutions (20 Units/mL) of r-FVIII proteins        representing the 4 additional haplotypic forms, H3, H4, H5 and        H6, of the wildtype human FVIII protein (i.e., r-hFVIII-H3,        r-hFVIII-H5 and r-hFVIII-H6).        13. immunodepleted FVIII-deficient plasma. Reconstitute each        vial of NoFact-VIIIi FVIII-deficient plasma with 1.0 mL        distilled water. Swirl gently and allow to stand for 20 minutes        at room temperature. Stable 8 hours at 2-6° C.

d) Controls

A. System-N (normal): reconstitute with 1 mL of reagent grade water. Letsit for 30 minutes. Mix well. DO NOT INVERT. Stable for 8 hours on theSTA.

B. System-P (abnormal): reconstitute in 1 mL of reagent grade water. Letsit 30 minutes. Mix well. DO NOT INVERT. Stable for 8 hours on the STA.System-N and -P are normal and abnormal controls that are set up to runautomatically at 8-hour intervals. These should be performed at leastonce per shift if testing is performed. System-N and -P must be run anytime that new reagent is made up. Control results are filed in theinstrument's QC file automatically. All results for a 24-hour periodwill be reduced to a “mean” at midnight. This mean is used in thestatistical data and is plotted on the Levy-Jennings chart.

C. Quality Control will automatically be run when a new calibrationcurve has been requested. If using a stored curve, the STA willautomatically run QC if the patient samples have been loaded. QC canalso be ordered manually before loading patient samples.

D. All control ranges are monitored automatically by the STA. If anycontrols are outside the ±2SD range, the STA will audibly and visuallyalarm the operator. Otherwise, the results can be found in the AUTOCONTROL file and the individual QC files. Control results areautomatically filed in the STA QC file. All results for a 24-hour periodwill be reduced to a “mean” value at midnight. This mean is used in thestatistical data and is plotted on the Levy-Jennings chart as a dailymean.

e) Procedure

A. Dilute patient plasma in 12×75 plastic tubes with imidazole bufferedsaline as follows:

TABLE 1 Patient plasma dilution series. Dilution Patient Plasma DilutionSeries 1:2 500 μL patient plasma + 500 μL imidazole buffered saline 1:4500 μL of 1:2 patient dilution + 500 μL imidazole buffered saline 1:8500 μL of 1:4 patient dilution + 500 μL imidazole buffered saline 1:16500 μL of 1:8 patient dilution + 500 μL imidazole buffered saline 1:32500 μL of 1:16 patient dilution + 500 μL imidazole buffered saline 1:64500 μL of 1:32 patient dilution + 500 μL imidazole buffered saline 1:128500 μL of 1:64 patient dilution + 500 μL imidazole buffered saline 1:256500 μL of 1:128 patient dilution + 500 μL imidazole buffered saline1:512 500 μL of 1:256 patient dilution + 500 μL imidazole bufferedsaline

B. To perform a haplotype-specific Bethesda assay, the followingpatient-specific medical history information must first be obtained ordetermined:

1. The wildtype FVIII background (H1, H2, H3, H4, H5, or H6) withinwhich a patient's F8 mutation arose, and

2. Which FVIII replacement product(s) the patient has been administered.Currently there are 3 structurally-distinct r-FVIII proteins that arecommercially available for clinical use in factor replacement therapy:(a) Kogenate (Bayer HealthCare, LLC; Berkeley, Calif.); (b) Recombinate(Baxter Healthcare Corporation; Westlake Village, Calif.); and (c)Refacto (Wyeth/Genetics Institute; St. Louis, Mich.). Kogenate is afull-length B-domain containing r-FVIII molecule that is identical atthe amino acid sequence level to the naturally-occurring wildtype formof human FVIII represented by haplotype H1. Recombinate is afull-length, B-domain containing r-FVIII molecule that is identical inamino acid sequence to the naturally-occurring wildtypeform of humanFVIII represented by haplotype H2. In contrast to Kogenate andRecombinate, Refacto is a non-naturally-occurring r-FVIII protein thatlacks the majority of the B-domain found in the human molecule. Kogenateand Recombinatevary at only 1 amino acid residue, position 1241, i.e.the site of a naturally-occurringbiallelic polymorphisms, D1241E, whichis encoded by 1 (C92714G) of the 4 nsSNPs found in the human F8. SinceRefacto lacks the B-domain, the location of D1241E, and has the sameallele as Recombinate and Kogenate at the three additional nsSNP sites(i.e., R484H, R776G and M2238V), are referred to as representing ahybrid haplotype designated H1/H2.

3. In microvials, make the necessary number of incubation mixes betweenimmuno-depleted FVIII-deficient human plasmas, which have first beenreconstituted to contain a homogenous population of r-FVIII moleculesthat represent only 1 of the 6 naturally-occurring forms of the wildtypehuman protein (i.e., H1, H2, H3, H4, H5, or H6), and a dilution seriesof the patient's plasma (see tables 2 & 3 below). For any given patient,this will require the preparation of at least one r-FVIII workingsolution, which contains the form of the protein represented by what thepatient has been treated with.

If, for example, a patient has only been treated with Kogenate (i.e.,H1) and hisbackground FVIII haplotype is H1, only 1 reconstituted plasmaworking solution is prepared (i.e., FVIII-deficient human plasmacontaining 1 U/mL of r-hFVIII-H1) and used to prepare the 11 incubationmixtures with the patient's plasma as shown in Table 2 below.

If, however, the background haplotype of a patient, who, like theprevious patient, has received only Kogenate, is H3, two reconstitutedplasma working solutions mustbe prepared: (a) FVIII-deficient humanplasma containing 1 U/mL of as before; and (b) and FVIII-deficient humanplasma containing 1 U/mL of r-hFVIIIH3 (to serve as a control). Testingin this patient requires that both of these FVIII working solutionswould be used to prepare the 11 incubation mixtures with the patient'splasma as shown in Tables 2 and 3 below. In this case, the mixture ofpatient's plasma and the FVIII molecule corresponding structurally towhat he has been treated with (r-hFVIII-H1), serves as the test mixture.

The test FVIII incubation mixtures for the patient discussed in theabove example, would be prepared, as shown in table X below, by mixing200 μL of the reagent FVIII-deficient human plasma (No-Fact'VIIIi)¹,that has first been reconstituted with r-hFVIII-H1 at 1 Unit/mL, witheither 200 μL of non-diluted patient's plasma or 200 μL of the patient'splasma that has first been diluted imidazole buffered saline (seeTable 1) as shown.

TABLE X Test incubation mixtures for 1^(st) & 2^(nd) example patientsdescribed above. Tube Test Incubation Mixtures (—)- 200 μL normal pooledplasma + 200 μL imidazole buffer saline control 1:1 200 μL human plasma(r-hFVIII-H1) + 200 μL patient's non- diluted plasma 1:2 200 μL humanplasma (r-hFVIII-H1) + 200 μL patient's 1:2 plasma dilution 1:4 200 μLhuman plasma (r-hFVIII-H1) + 200 μL patient's 1:4 plasma dilution 1:8200 μL human plasma (r-hFVIII-H1) + 200 μL patient's 1:8 plasma dilution1:16 200 μL human plasma (r-hFVIII-H1) + 200 μL patient's 1:16 plasmadilution 1:32 200 μL human plasma (r-hFVIII-H1) + 200 μL patient's 1:32plasma dilution 1:64 200 μL human plasma (r-hFVIII-H1) + 200 μLpatient's 1:64 plasma dilution 1:128 200 μL human plasma (r-hFVIII-H1) +200 μL patient's 1:128 plasma dilution 1:256 200 μL human plasma(r-hFVIII-H1) + 200 μL patient's 1:256 plasma dilution 1:512 200 μLhuman plasma (r-hFVIII-H1) + 200 μL patient's 1:512 plasma dilution ¹No-FactVIIIi is generated by FVIII-immunodepletion of pooled normalhuman plasma

TABLE Y Control incubation mixtures for 2^(nd) example patient describedabove. Tube Control Incubation Mixtures (—)- 200 μL normal pooledplasma + 200 μL imidazole buffer saline control 1:1 200 μL human plasma(r-hFVIII-H3) + 200 μL patient's non- diluted plasma 1:2 200 μL humanplasma (r-hFVIII-H3) + 200 μL patient's 1:2 plasma dilution 1:4 200 μLhuman plasma (r-hFVIII-H3) + 200 μL patient's 1:4 plasma dilution 1:8200 μL human plasma (r-hFVIII-H3) + 200 μL patient's 1:8 plasma dilution1:16 200 μL human plasma (r-hFVIII-H3) + 200 μL patient's 1:16 plasmadilution 1:32 200 μL human plasma (r-hFVIII-H3) + 200 μL patient's 1:32plasma dilution 1:64 200 μL human plasma (r-hFVIII-H3) + 200 μLpatient's 1:64 plasma dilution 1:128 200 μL human plasma (r-hFVIII-H3) +200 μL patient's 1:128 plasma dilution 1:256 200 μL human plasma(r-hFVIII-H3) + 200 μL patient's 1:256 plasma dilution 1:512 200 μLhuman plasma (r-hFVIII-H3) + 200 μL patient's 1:512 plasma dilution

C. Mix the incubation mixtures, cap vials, place in the 37° C. waterbathand incubate for 2 hours.

D. At the end of 2 hours, perform the FVIII assay on the control tubes,the tube with the 1:1 dilution and the tube with the 1:2 dilution, onthe STA as follows:

1. Refer to the START-UP procedure for the STA before running patientsamples on the STA at the start of each shift.

2. Request quality control if using a stored curve: through the MAINMENU under CALM/CONTROL select QUALITY CONTROL and enter. Cursor to theselected factor assay(s) and select by pressing F1 and then F10 to runthe QC.

3. Load patient samples: access the sample drawer(s) through the MAINMENU under LOADING. After the drawer opens, identify the type ofspecimen, such as micro sample, with F8, or STAT, with F12. Identify thesample by bar-coding or manually entering on the keyboard the patientidentification number and then placing the sample into the drawer.

4. In MANUAL MODE, the operator must order the test from the menu.Select the factor that has a ×10 dilution. Press F10 to save.

5. As soon as the sample drawer closes, the TEST STATUS screen willappear. If there is not enough reagent(s) to run the test, the suspectreagent(s) will appear in red with the amount of deficiency. Thisdeficiency will BLOCK the sample pipettor. When this occurs, add thedeficient reagent(s) to run the samples.

6. All dilutions of the calibrator, controls and patient samples areautomatically prepared by the STA according to the parameters entered inthe Test Set-up. If the patient results fall outside the assay range,the STA automatically re-tests the sample in question at an appropriatedilution provided that the option has been entered in the Test Set-up.

7. All patient results are displayed on the TEST PANEL screen andautomatically print out and transmit if selected.

8. For results in question that need operator intervention, cursor tothe identification number in the TEST PANEL screen and enter. This willbring up the FILE PROCESSING screen. Follow the options in the left-handcorner of the screen, i.e. re-run test.

f) Procedural Notes

A. Calculate the residual FVIII:C activity of each patient dilutionincubation mixture by using the following calculations:

${{Residual}\mspace{14mu} {FVIII}\text{:}\mspace{14mu} C\mspace{14mu} {activity}} = {\frac{\begin{matrix}{{Factor}\mspace{14mu} {VIII}\text{:}} \\{C\mspace{14mu} {Activity}\mspace{14mu} ({Patient})}\end{matrix}}{\begin{matrix}{{Factor}\mspace{14mu} {VIII}\text{:}} \\{C\mspace{14mu} {Activity}\mspace{14mu} ({Control})}\end{matrix}} \times 100}$

B. If the residual FVIII:C activity level is <75%, the residual FVIII:Cactivity is converted to a Bethesda unit factor using the followingchart and calculation:

Residual FVIII:C Level Bethesda Units Bethesda Factor 75% 0.40 Chart:73% 0.45 70% 0.50 68% 0.55 66% 0.60 64% 0.65 61% 0.70 59% 0.75 57% 0.8055% 0.85 53% 0.90 51% 0.95 50% 1.00 48% 1.05 46% 1.10 45% 1.15 43% 1.2042% 1.25 41% 1.30 40% 1.35 38% 1.40 37% 1.45 35% 1.50 34% 1.55 33% 1.6032% 1.65 30% 1.70 29% 1.75 28% 1.80 27% 1.85 26% 1.90 25% 2:00

C. If the residual FVIII:C activity is >75% of the control value in the1:1 dilution tube and the 1:1 dilution tube, report out <1.0 Bethesdaunit.

D. Perform the following calculation to determine Bethesda units/mLplasma of inhibitor present using the residual FVIII:C value closest to50% of the control value.

Bethesda units×Dilution factor from chart=FVIII:C inhibitor in factorfrom chart of patient plasma used to Bethesda units/mL determineresidual FVIII:C plasma  1. Calculation:

DILUTION FACTOR CHART Dilution of Patient Plasma Dilution Factor 1:1 11:2 2 1:4 4 1:8 8 1:16 16 1:32 32 1:64 64 1:128 128 1:256 256 1:512 512

2. Example:

Control FVIII:C level=50%Patient FVIII:C level on 1:4 dilution=25%

${{Residual}\mspace{14mu} {FVIII}\mspace{14mu} {level}} = {{\frac{25}{50} \times 100\%} = {50\%}}$

50% in Bethesda units (BU) from Bethesda factor chart (chart B)=1.0 BU1.0 BU×4 (patient dilution)=4.0 BU (final result)

E. If the residual FVIII:C activity falls below 25%, the test must berepeated using higher dilutions of patient plasma until at least onevalue of greater than 25%, residual FVIII:C activity is obtained.

F. If more than one dilution falls between the 25% and 75% residualFVIII:C activity range, the final result should be the average of allthese results.

G. To search for weak inhibitors, a >ratio of patient to pooled normalplasma can be used in the incubation mixture. If 1 part pooled normalplasma and one part patient plasma do not give a satisfactory result,increase the amount of patient plasma to pooled normal plasma: add 0.5mL patient plasma to 0.1 mL pooled normal plasma and incubate 2 hours at37° C. At the end of the 2 hour incubation, perform a FVIII:C assay onthe specimen and calculate the %-residual activity as before. Determinethe Bethesda unit as before. Divide the Bethesda units obtained by 5 toobtain the Bethesda units actually present in the patient plasma.

g) Result Reporting

A. If residual FVIII:C activity level is greater than 75%, report as: Noinhibitor present (Bethesda units <1.0).

B. If residual FVIII:C activity level is 75% or less, report as:inhibitor present (Bethesda units/mL of plasma).

h) Interpretation

One Bethesda unit is described as the concentration of an antibody whichproduces a residual FVIII:C of 50%, in a precisely controlled incubationmixture that is maintained at 37° C. for 2 hours. It is stated as 1 unitof inhibitor/mL of plasma. This method is primarily intended for use inmeasuring inhibitors that occur in hemophiliacs. Very weak inhibitorsmay not be detected by this method. Nearly all patients in whominhibitors develop have severe hemophilia A (FVIII:C of <1%). In mostpatients, the antibody persists for years and there is an anamnesticresponse after transfusion. As the inhibitor titer increases, thepatient usually becomes less responsive to replacement therapy. In somepatients, the antibody titer remains low and does not increase afterreplacement therapy. FVIII:C inhibitors occasionally develop innon-hemophiliacs who have previously received transfusions. If weakinhibitors can be detected and quantitated, then the course of suchinhibitors can be evaluated and related to such factors as blood producttherapy. In some patients, inhibitor strength progress' from the barelydetectable to the potent. Some patients with potent inhibitors who arenot exposed further to blood products may have barely detectableinhibitors later.

The following references are herein incorporated by reference in theirentireties:

A. Children's Hospital Medical Center, Los Angeles, Calif.

B. Kasper, Carol K. and Pool, Judith G., Measurement of mild inhibitorsin Bethesda Units. Thrombosis et. Diathesis: Haemorrhagia. 1975, Vol.34, p. 875.

C. Kasper, Carol K. and Pool, Judith G., et al. A More UniformMeasurement of Factor VIII Inhibitors. Thrombosis et. Diathesis:Haemorrhagica. 1975 Vol. 34, p. 869.

D. Thomson, Jean M., ed. Blood Coagulation and Haemostasis: A PracticalGuide. 2nd ed., Edinburgh, Great Britain, 1980, pp. 82-82.

E. Triplett, Douglas A., ed. Laboratory Evaluation of Coagulation. 1sted., Chicago, Ill., 1981, pp. 128-129.

F. Triplett, Douglas A., and Harms, Cathy S. Procedures for theCoagulation Laboratory. Chicago, Ill., 1981, pp. 71-75.

G. Lusher, J., Abildgaard, C., Arkin, S., Mannucci, P. M., Zimmermann,R., Schwartz, L., Hurst, D. Human recombinant DNA-derived antihemophilicfactor in the treatment of previously untreated patients with hemophiliaA: final report on a hallmark clinical investigation. J. Thromb.Haemost. 2(4):574-83, 2004.

H. Lindgren, A., Wadenvik, H., and Tengborn, L. Characterization ofinhibitors to FVIII with an ELISA in congenital and acquired haemophiliaA. Haemophilia. 8(5):644-648, 2004.

4. Methods of Genetic Testing

Rapid-cycle polymerase chain reaction (PCR) with an allele-specificfluorescent probe can be used for SNP genotyping. High-resolutionamplicon melting curve analysis can be used for SNP genotyping.Fluorescent resonance energy transfer (FRET) hybridization probes fordetection of the base changes (Lyon Molecular Diagnosis 1998 3:203,herein incorporated by reference) can also be used for SNP genotyping.

a) Lightcycler

(1) Method

A method for determining a subject haplotype can combine a rapid-cyclepolymerase chain reaction (PCR) with an allele-specific fluorescentprobe melting for mutation detection. This method combined with rapidDNA extraction, can generally provide results within 60 min afterreceiving a blood sample. This method allows for easy, reliable, andrapid detection of a polymorphism, and is suitable for typing both smalland large numbers of DNA samples.

The LightCycler® system enables the detection of single nucleotidepolymorphisms. It combines PCR amplification and detection into a singlestep. The platform enables the real-time detection of a specific PCRproduct followed by melting curve analysis of hybridization probes. Thetechnology is based on the detection of two adjacent oligonucleotideprobes, whose fluorescent labels communicate through fluorescenceresonance energy transfer (FRET). The molecular concept of singlenucleotide polymorphism (SNP) detection is as follows: one of the probesserves as a tightly bound anchor probe and the adjacent sensor probespans the region of sequence variation. During the melting of the finalPCR product, the sequence alteration is detected as a change in themelting temperature of the sensor probe. For a typical homozygous wildtype sample, a single melting peak is observed; for mixed alleles, twopeaks are observed; and for a homozygous mutated sample, a single peakat a temperature different from the wild type allele is observed. Thetemperature shift induced by one mismatched base is usually between 5and 9° C. and easily observable.

The FVIII haplotyping assay allows the rapid detection and genotyping offour non-synonymous single nucleotide polymorphisms (nsSNPs) of the (Gto A at cDNA position 1508, A to G at cDNA position 2383, G to C at cDNAposition 3780, and A to G at cDNA position 6769), from DNA isolated fromhuman whole peripheral blood. The test can be performed on theLightCycler® Instrument utilizing polymerase chain reaction (PCR) forthe amplification of F8 DNA recovered from clinical samples andfluorigenic target-specific hybridization for the detection andgenotyping of the amplified F8 DNA. Disclosed are oligonucleotidescomprising cDNA sequences for each haplotype: H1 (SEQ ID NO: 7), H2 (SEQID NO: 8), H3 (SEQ ID NO: 9), H4 (SEQ ID NO: 10), H5 (SEQ ID NO: 11), H6(SEQ ID NO: 12).

The FVIII haplotyping test is an in vitro diagnostic test for thedetection and genotyping of four non-synonymous human F8 SNPs—which havecommon minor alleles conferring the amino acid substitutions R484H,R776G, E1241D and M2238V—whose naturally-occurring allelic combinations(i.e., haplotypes H1, H2, H3, H4, H5, and H6) encode six structurallydistinct wildtype forms of the human FVIII protein. Because only two ofthese haplotypes, H1 and H2, are represented by the currently availablerecombinantl and plasma-derived2 FVIII preparations used clinically, theFVIII haplotyping test will aid physicians select matched3 FVIIIreplacement products that reduce the frequency at which their hemophiliaA patients develop FVIII inhibitors and immunologic refractoriness toreplacement therapy.

Use of the FVIII haplotyping test as a component assay in laboratoryalgorithms for thrombophilia evaluation, can improve the diagnosticaccuracy of thrombosis risk assessment, since the findings of recentgenetic studies have demonstrated that the alleles of at least one ofthe these four nsSNPs F8 (i.e., D1241E) are functionally distinct andinfluence circulating FVIII levels. For example, because individualswith the E-allele (protein level; or G at the nucleotide level), werefound to have an ˜25% lower mean circulating FVIII level, than thosewith the more prevalent D-allele (protein level; or C at the nucleotidelevel), this SNP is a determinant of thrombosis risk at the populationlevel. The test is performed on the LightCycler® Instrument (In the EU:serial number 2021 to 5602) using SW 3.5. The sample preparation isperformed according to the procedure described below.

The development of neutralizing alloantibodies to replacementcoagulation factor (F)VIII is a serious complication in the managementof hemophilia A. The pathogenesis of these antibodies, termedinhibitors, is complex and incompletely understood. Through DNAsequencing, four common nonsynonymous single nucleotide polymorphisms(nsSNPs) were identified in the FVIII genes (F8) from 137 unrelatedhealthy people representing seven ethnic groups and identified. Thenaturally-occurring allelic combinations of these four nsSNPs werefurther found to encode six structurally-distinct wildtype FVIIIproteins (haplotypes). This finding that FVIII is not monomorphic in manraises the possibility that structural differences between thebackground haplotype of a patient's endogenous molecule and the infusedwildtype replacement FVIII protein(s) may contribute to the developmentof inhibitors.

African American (AA) hemophilia A patients experience a two-foldgreater incidence of inhibitors in comparison to Caucasians. AfricanAmericans express five haplotypes of FVIII, designated H1, H2, H3, H4and H5. In contrast, Caucasians express only H1 and H2. The recombinantFVIII molecules in replacement products used clinically have sequenceidentity with H1 and H2. Similarly, plasma-derived FVIII preparationsmarketed in the US are essentially highly enriched with the H1 and H2forms of the protein, because the blood donor population ispredominantly Caucasian. The H4 haplotype, expressed by ˜4% of AfricanAmericans, is defined by the nsSNP R484H, which is located in animmunodominant epitope within the A2-domain. Two other haplotypes, H3and H5, which together are expressed in ˜23% of African Americans, havethe minor allele of M2238V, a nsSNP in the C2-domain immunodominantepitope. These findings indicate that >25% of African Americanhemophilia A patients are treated with replacement products containingFVIII molecules which vary from their own at the hemophilic mutationsite, and at one or more additional sites, with one always located in andominant inhibitory epitope.

The use of this assay can guide hemophilia treaters in selecting FVIIIreplacement products that are matched as closely as possible to a givenpatient's endogenous background haplotype, which he will presumably havetolerance to, in order to reduce the frequency of inhibitor development.

Four fragments of the FVIII gene, referred to as amplicon-1 (189-bp), -2(161-bp), -3 (151-bp), and -4 (163-bp), are individually amplified inseparate PCRs from human genomic DNA using the four pairs of F8 specificprimers listed above.

The amplicons, each of which contain one of the four F8 nsSNPs, aredetected by fluorescence using a specific pair of hybridization probes(SEQ ID NOS: 27 & 28 for amplicon 1; SEQ ID NOS: 31 & 32 for amplicon 2;SEQ ID NOS: 35 & 36 for amplicon 3; and SEQ ID NOS: 39 & 40 for amplicon4). The paired hybridization probes comprise two differentoligonucleotides that hybridize to an internal sequence of the amplifiedF8 fragment during the annealing phase of the PCR cycle. One probe islabeled at the 5′-end with LightCycler® Red 640, and to avoid extension,modified at the 3′-end by phosphorylation. The other probe is labeled atthe 3′-end with fluorescein.

Only after hybridization to the template DNA, do the two probes come inclose proximity, resulting in fluorescence resonance energy transfer(FRET) between the two fluorophores. During FRET, fluorescein, the donorfluorophore, is excited by the light source of the LightCycler®Instrument, and part of the excitation energy is transferred toLightCycler® Red 640, the acceptor fluorophore.

The emitted fluorescence of LightCycler® Red 640 is then measured by theLightCycler® Instrument.

Genotyping: The paired hybridization probes are also used to determinethe nsSNP genotype contained within the amplicon by performing a meltingcurve analysis after the amplification cycles are completed and theamplicon is present at increased concentration.

In amplicons 2 and 4, the LightCycler® Red 640-labeled hybridizationprobe hybridizes to a part of the target sequence that is notpolymorphic and functions as an anchor probe. In amplicons 1 and 3, theLightCycler® Red 640-labeled hybridization probe hybridizes to a part ofthe target sequence that spans the SNP site (SNP probe).

The Fluorescein-labeled hybridization probe hybridizes to a part of thetarget sequence in amplicons 2 and 4 that is polymorphic and functionsas the SNP probe. In amplicons 1 and 3, it hybridizes to a part of thetarget sequence that is not polymorphic and functions as an anchorprobe.

During the melting curve analysis of amplicons 1 and 3, increasingtemperature causes the fluorescence to decrease because the LightCycler®Red 640-labeled hybridization probe, the shorter of the two probes (SNPprobe) dissociates first and the two fluorescent dyes are no longer inclose proximity. During the melting curve analysis of amplicons 2 and 4,in contrast, the increasing temperature causes the fluorescence todecrease because the Fluorescein-labeled hybridization probe, theshorter of the two probes (SNP probe) dissociates first and the twofluorescent dyes are no longer in close proximity.

For both amplicon 2 and 4, if the more frequent A-allele is present atthe site of nsSNP #2 (A2383G [R776G]) or nsSNP #4 (A6769G [M2238V]),respectively, the 0.0 mismatch of the SNP probe with the target sequencedestabilizes the hybrid so the decrease in fluorescence will occur at alower temperature. For amplicon 1, if the less frequent A-allele ispresent at the nsSNP #1 site (G1508A [R484H]), the mismatch of the SNPprobe with the target sequence destabilizes the hybrid so the decreasein fluorescence will occur at a lower temperature. Likewise, foramplicon 3, if the less frequent G-allele4 is present at the nsSNP #3site (C3780G [D1241E]), the mismatch of the SNP probe with the targetsequence destabilizes the hybrid so the decrease in fluorescence willoccur at a lower temperature. With the less frequent G-allele present atthe site of either nsSNP #2 (amplicon 2) or nsSNP #4 (amplicon 4), orthe more frequent G- or C-alleles at the site of either nsSNP #1(ampliconl) or nsSNP #3 (amplicon 3), the genotype mismatches will notoccur, and therefore the heteroduplex has a higher melting temperature(Tm). The heterozygous genotype at all four nsSNPs exhibits adistinctive combination of properties.

(2) Primers/Probes

Disclosed herein are four sets of oligonucleotide sequences (i.e., eachset contains a PCR primer set and a FRET probe set) used as the reagentsin the LightCycler/Hybridization Probe Analysis-based assay to rapidlygenotype the four nsSNPs in the F8 gene of a given individual, andtherefore to define their FVIII haplotype. These oligonucleotides (i.e.,both primers and probes) are named according to the nucleotide positioncorresponding to the major transcriptional start of the F8 gene, foundin SEQ ID NO:1, as well as the five other F8 gene sequences (i.e., SEQID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6), whichcorresponds to nucleotide position 1001. This site (i.e., nucleotideposition 1001 in the F8 gene sequences) can also be correlated with thea single precise nucleotide position within the GenBank human F8reference sequence (accession number is NG_(—)005114). The human F8transcriptional start site sequence corresponds precisely withnucleotide position 465,469 in the complementary strand to thatcontained within NG_(—)005114.

(a) nsSNP #1: (G61620A/G1508A/R484H)

(SEQ ID NO: 25) H-F8-61568-F (forward): 5′-AATCAAGCAAGCAGACCAT (SEQ IDNO: 26) H-F8-61756-R (reverse): 5′-GGCCAACAGCTGGAGAA (SEQ ID NO: 27)H-F8-61629 (probe 1):5′-LC^(Red640)-TACAAAGGACGGACATCAGTGATTCCG-phosphate (SEQ ID NO: 28)H-F8-61666 (probe 2): 5′-ATCGAGGGAATATTTACCTTTTGGTAATCTCCTTG-Fluorescein

(b) nsSNP #2: (A91317G/A2383G/R776G)

(SEQ ID NO: 29) H-F8-91221-F (forward): 5′-CAGAATTCAAGACACCCTAGC (SEQ IDNO: 30) H-F8-91381-R (reverse): 5′-CTCTGTCGCAAGAGCATC (SEQ ID NO: 31)H-F8-91298 (probe 1):5′-LC^(Red640)-AGTCTTCTCTATGTCATTTTCTGGAATTGTGGTGG- phosphate (SEQ IDNO: 32) H-F8-91320 (probe 2): 5′-TTCCGTGTGCAAACCAAGGG-Fluorescein

(c) nsSNP #3: (C92714G/C3780G/D1241E)

(SEQ ID NO: 33) H-F8-92624-F (forward): 5′-GCCTCAGATACATACAGTGAC (SEQ IDNO: 34) H-F8-92774-R (reverse): 5′-TGTTCTATTTGTTGAATCATTTAATGACC (SEQ IDNO: 35) H-F8-92670 (probe 1):5′-CTTTTCTTACTOAGCACTAGGCAAAATGTAGAAGG-Fluorescein (SEQ ID NO: 36)H-F8-92707 (probe 2): 5′-LC^(Red640)-CATATGACGGGGCATATGCTCCA-phosphate

(d) nsSNP #4: (A162161G/A6769G/M2238V)

(SEQ ID NO: 37) H-F8-162073-F (forward): 5′-AATGGTGACCAAGAGGC (SEQ IDNO: 38) H-F8-162235-R (reverse): 5′-GATGAGGAACTCCTTCACATAC (SEQ ID NO:39) H-F8-162136 (probe 1):5′-LC^(Red640)-CCACTCTTTTGGATTATTCACCTGAGGGCAATAGA- phospate (SEQ ID NO:40) H-F8-162166 (probe 2): 5′-TTTCACTGTCTTCTGGAAGTCCACTTGC-Fluorescein

-   b) High-Resolution Amplicon Melting Curve Analysis

Disclosed are methods for determining a subject haplotype utilizinghigh-resolution amplicon melting curve analysis.

(1) Method

There are many methods available to genotype SNPs. Available methodsthat require a separation step include restriction fragment lengthpolymorphism analysis, single-nucleotide extension, oligonucleotideligation, and sequencing.

Homogeneous, closed-tube methods for SNP genotyping that do not requirea separation step are attractive for their simplicity and containment ofamplified products. Most of these methods are based on PCR. Meltingcurve analysis in conjunction with real-time PCR was introduced in 1997(Ririe Anal. Biochem 1997 245:154), incorporated herein by reference.The ds DNA dye SYBR Green I was used for amplicon melting analysis andprovided a rough characterization of what was amplified. Subsequently,hybridization probes were used to interrogate a limited region of theamplicon for sequence differences by melting curve analysis (Lay ClinChem 1997 43:2262), incorporated herein by reference. The primaryadvantage of this method or other real-time PCR methods is that it caninterrogate the entire region under the probe, rather than justdetecting the presence or absence or a specific allele. Twohybridization probes are most commonly used for melting curve analysisof small sequence variations.

If PCR is done with a 5′-labeled primer, high resolution meltinganalysis can distinguish different homozygotes and heterozygotes (GundryClin Chem 2003 49:396), incorporated herein by reference. Thedisadvantages of this method are the requirements of a labeled primerand that the sequence variations must be within the melting domain thatincludes the labeled primer.

SYBR Green I has limitations for detection of small sequence variations.Detection of heterozygotes by melting curve analysis with SYBR Green Ihas been possible only when extra steps are added between amplificationand analysis, such as amplicon purification and addition of high amountsof the dye (Lipsky Clin Chem 2001 47:635), incorporated herein byreference, or urea (Elenitoba-Johnson Am J Pathol 2001 159:845),incorporated herein by reference. Wittwer et al (Clin Chem 2003 49:6),incorporated herein by reference, screened several ds DNA dyes and foundone, LCGreen I, that did detect heterozygotes, but did not inhibit orotherwise adversely affect PCR amplification. Unlike SYBR Green I, LCGreen I saturates PCR products without inhibiting amplification and doesnot redistribute as the amplicon melts.

High resolution melting of PCR amplicons with LCGreen I was recentlyintroduced as a homogeneous, closed-tube method for rapid SNP genotypingthat does not require the use of probes. Genomic DNA is amplified in thepresence of LCGreen I in LightCycler (Roche Applied Science) capillarytube. The capillary tube is then transferred to a high-resolutionmelting instrument'(1R-1, Idaho Technology) for the melting curveacquisition and analysis. The instrument surrounds the capillary tubewith an aluminum cylinder that is heated by a coil wound around theoutside. Sample temperature and fluorescence signals are converted to16-bit digital signals, providing resolution down to 0.002° C. and0.002% normalized fluorescence. Approximately 50 data points areacquired for every 1° C. The melting curve acquisition requires only 1to 2 min. Melting data are analyzed with the HR-1 instrument software.Fluorescence values are normalized between 0% and 100% by first defininglinear baselines before and after the melting transition of each sample.Within each sample, the fluorescence of each acquisition is calculatedas the percentage of fluorescence between the top and bottom baselinesat each acquisition temperature. Heterozygotes are identified by achange in melting curve shape, and different homozygotes aredistinguished by a change in melting temperature (Tm).

When coupled with rapid cycle PCR, SNP genotyping can be performed in 30minutes or less. Although LightCycler real-time PCR instruments arecommonly used in research and molecular diagnostic laboratories formelting curve analysis, the resolution of real-time PCR instruments islimited and they cannot distinguish the small Tm differences betweenhomozygotes. Dedicated melting instruments have recently become 0.0available. Although only one sample at a time is analyzed the analysistime is so short (1 to 2 min) that the throughput is relatively high.

High-resolution melting of small PCR amplicons (<50 bp) is simple,rapid, and inexpensive method for SNP genotyping. Engineered plasmidsrepresenting all of the possible SNP base changes, and samplescontaining the medically important factor V (Leiden) 1691G>A,prothrombin 20210G>A, methylenetetrahydrofolate reductase 1298A>C,hemochromatosis 187C>G, and β-globin (hemoglobin S) 17A>T weresuccessfully genotyped using this method (Liew Clin Chem 2004 50:7),incorporated herein by reference. In all cases, heterozygotes wereeasily identified because the heteroduplexes altered the shape of themelting curves. Approximately 84% of human SNPs involve a base exchangebetween A:T and G:C base pairs (Venter Science 2001 291:1304), and thehomozygotes are easily genotyped by Tms that differ by 0.8 to 1.4° C.However in the remaining SNPs, the bases only switch strands andpreserve the base pair, producing very small Tm differences betweenhomozygotes (<0.4° C.). Although most of these cases can still begenotyped by Tm, about a quarter have nearest neighbor symmetry(complementary bases), and the homozygotes cannot be distinguished. Inthese cases adding a known homozygous genotype to unknown samples allowsmelting curve separation of all three genotypes. This method was used toidentify C/C and G/G homozygotes in the hemachromatosis 187C>G SNPgenotyping assay mentioned above (Liew Clin Chem 2004). This representsthe worst case scenario, since the nearest neighbors of this SNP werecomplementary bases (A and T). When the bases flanking the SNP aresymmetrical, the nearest neighbor stability calculations are nearlyidentical.

Disclosed herein is the use of high-resolution melting curve analysis inassays for the four F8 nsSNPs (G61620A, G92714C, A162161G & A91317G)whose naturally occurring allelic combinations (haplotypes) encode the 6different wildtype forms of FVIII in humans.

Correlation of nsSNP genotypes with FVIII haplotype 1 2 3 4 nsSNP no.(G61620A) (A91317G) (G92714C) (A162161G) Haplotype 1 G A C A Haplotype 2G A G A Haplotype 3 G A G G Haplotype 4 A A G A Haplotype 5 G A C GHaplotype 6 G G G A

DNA from whole blood samples can be extracted using a MagNA Pureinstrument with a total nucleic acid kit. As Tm is strongly dependent onthe ionic strength, it is important that samples are extracted in thesame way and end up in the same buffer. Using this automated extractionsystem ensures uniformity in technique and nucleic acid yield.

To maximize the Tm differences between the genotypes, the amplicons canbe made as short as possible (<50 bp). The sequence informationsurrounding the SNP will can be entered into computer software(“SNPWizard” at http:\\DNAWizards.path.utah.edu). The 3′ end of eachprimer can be placed immediately adjacent to the SNP and its lengthincreased in its 5′ direction until its predicted Tm is as close to55-60° C. as possible. The primer pair can then be checked for thepotential to form primer dimers or other non-specific amplicons. Ifalternative products are likely, then the 3′ end of one primer can beshifted one base away from the SNP and the process can be repeated untilan acceptable pair is found. Candidate primer pairs can be synthesizedusing standard phosphoramidite chemistry.

Separate PCRs for each SNP can be performed in 10 μl volumes with thefollowing reaction constituents: dNTPs (200 μM), KlenTaq1 polymerase(0.4 U), TaqStart antibody (88 ng), forward and reverse primers (250 nMeach), LC Green I (1×), MgCl2 (3 mM), Tris buffer pH 8.3 (50 mM), and 1μl DNA sample. Thermal cycling can be performed in a LightCyclerreal-time PCR instrument. Rapid thermal cycling can be performed between85° C. and the annealing temperature at the programmed transition rateof 20° C./sec. Amplified samples can be heated to 94° C. in theLightCycler and rapidly cooled to 40° C. The LightCycler capillaries canthen be transferred to the BR-1 instrument and heated at 0.3° C./sec.Samples can be analyzed between 65 and 85° C. with the instrumentsoftware. Fluorescence vs. temperature plots can be normalized asdescribed above.

The ability of the high-resolution melting analysis to correctlyidentify the 4 informative SNPs and assign 6 Factor VIII haplotypes canbe assessed with a set of samples authenticated by nucleic acidsequencing.

High-resolution melting can correctly identify SNP genotypes 1, 2, and 4since they all represent transitions of G and A. High-resolution meltingcan correctly identify SNP genotype 3 as the G and C is simply inverted,that is, the bases switch strands but the base-pair remains the same.Differences in amplicon Tm still result from different nearest-neighborinteractions but they are usually small. However, SNP 3 is flanked bynoncomplementary bases (A and G) and results in small butdistinguishable change in Tm for the amplicon containing base inversion.

(2) Primers

Four oligonucleotide primer pairs (i.e., 8 total oligonucleotides)required to utilizing the method known as “High-Resolution AmpliconMelting-Curve Analysis” to genotype the 4 nsSNPs in the FVIII gene (F8)whose naturally-occurring allelic combinations encode the 6 distinctwildtype forms of the FVIII protein in humans.) The following four pairsof oligonucleotide sequences that represent the four primer pairs usedin the PCR/High-Resolution Amplicon Melting-Curve-Analysis-based assayenable rapid genotyping of the 4 nsSNPs in the F8 gene of a givenindividual, and therefore to define their FVIII haplotype. Theseoligonucleotides are named according to the nucleotide positioncorresponding to the major transcriptional start of the F8 gene, whichcorresponds to nucleotide position 1001 of SEQ ID NO:1, as well as tonucleotide position 1001 in the five other F8 gene sequences (i.e., SEQID NO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6). This site(i.e., nucleotide position 1001 in the F8 gene sequences) can also becorrelated with the a single precise nucleotide position within theGenBank human F8 reference seqeunce (accession number is NG_(—)005114).The human F8 transcriptional start site sequence corresponds preciselywith nucleotide position 465,469 in the complementary strand to thatcontained within NG_(—)005114.

(a) nsSNP #1

G61620A (R484H): the minor allele of this nsSNP (i.e., which is an “A”at the nucleotide level and a “histidine” [H] at the amino acid level)is restricted to African-Americans.

A61620 (R484H)

H-F8-61599-F (forward primer): 5′-CTCACGGAATCACTGATGTC (SEQ ID NO: 41)H-F8-61643-R (reverse primer): 5′-GTAATCTCCTTGAATACAAAGG (SEQ ID NO: 42)nsSNP#1 forward genotyping primer Melting temperature (Tm): 40+20=60° C.nsSNP#1 reverse genotyping primer Melting temperature (Tm): 40+20=60° C.

(b) nsSNP #2

A91317G (R776G): the minor allele of this nsSNP (i.e., which is a “G” atthe nucleotide level and a “Glycine” [G] at the amino acid level) isrestricted to Chinese individuals

G91317 (R776G)

H-F8-91298-F (forward primer): 5′-TGACCCTTGGTTTGCACAC (SEQ ID NO: 43)H-F8-91339-R (reverse primer): 5′-TGTATTTTAGGCATAGGTGTTC (SEQ ID NO: 44)nsSNP#2 forward genotyping primer Melting temperature (Tm): 40+18=58° C.nsSNP#2 reverse genotyping primer Melting temperature (Tm): 32+28=60° C.

(c) nsSNP #3

G92714C (E1241D): the minor allele of this nsSNP (i.e., which is a “C”at the nucleotide level and a “aspartate” [D] at the amino acid level)is not restricted ethnically (Note that while C is the minor allele inthe African-American population, in all other human populations,including Caucasians, it is the opposite with the “G” nucleotide and“Glutamate” [B] being the minor allele).

C92714 (E1241D)

H-F8-92691-F (forward primer): 5′-CAAAATGTAGAAGGTTCATATGA (SEQ ID NO:45) H-F8-92738-R (reverse primer): 5′-TTGAAGTACTGGAGCATATGC (SEQ ID NO:46)nsSNP#3 forward genotyping primer Melting temperature (Tm): 28+32=60° C.nsSNP#3 reverse genotyping primer Melting temperature (Tm): 36+24=60° C.

(d) nsSNP #4

A162161G (M2238V): the minor allele of this nsSNP (i.e., which is a “G”at the nucleotide level and a “valine” [V] at the amino acid level) isrestricted to African-Americans.

G162161 (M2238V)

H-F8-162141-F (forward primer): 5′-AAGTGGACTTCCAGAAGACA (SEQ ID NO: 47)H-F8-162182-R (reverse primer): 5′-TAGTTACTCCTGTGACTTTCA (SEQ ID NO: 48)nsSNP#4 forward genotyping primer Melting temperature (Tm): 36+22=58° C.nsSNP#4 reverse genotyping primer Melting temperature (Tm): 32+26=58° C.

c) Functional Scan

(1) Method

See Example 2 for a discussion of an exemplary functional vacation scan.

(2) Primers

Disclosed herein are 39 pairs of oligonucleotides used for scanning thefunctional regions of the human F8 gene in order to findnaturally-occurring sequence variation that are candidates to havefunctionally distinct alleles that can influence thrombosis risk (forexample, the D1241E nsSNP, because the D-allele is associated with asignificantly higher mean FVIII levels than the E-allele) (See Example2, below). However because it also changes an amino acid in a proteinthat is used as a therapeutic replacement molecule that is naturallyimmunogenic, they can also influence the immune response to treatmentdepending upon which allele a patient has and which allele they aretreated with.

The SEQ ID's of the F8 gene sequences disclosed are used as the basis toname and thus define the exact position of the oligonucleotide sequencesbelow, which as mentioned above, are the 39 primer pairs necessary forboth amplifying and seqeuncing the F8 gene.

The nucleotide numbering scheme used for the F8 gene described in SEQ IDNo's 1-6 (and therefore for the oligonucleotides) is based on the majortranscriptional start of the F8 gene (i.e., the 5′-end of thepredominant mRNA species for transcribed from the F8 gene), whichcorresponds to nucleotide position 1001 of SEQ ID NO:1, as well as tonucleotide position 1001 in the 5 other F8 gene sequences (i.e., SEQ IDNO:2; SEQ ID NO:3; SEQ ID NO:4; SEQ ID NO:5; SEQ ID NO:6). This site(i.e., the transcriptional start or nucleotide position 1001 in the F8gene sequences) can also be correlated with the a single precisenucleotide position within the GenBank reference sequence for the humanF8 gene whose accession number is NG_(—)005114. Since the sequencecontained within NG_(—)005114 represents the antisense strand of the F8gene, the transcriptional start site corresponds precisely withnucleotide position 465,469 in the complementary strand to thatcontained within NG_(—)005114.

Ampli- Primer Name: Sequence: con: hF8-(−1214)-F5′-TTATCAAAGGGGCTTCTTGC-3′ A01 (SEQ ID NO: 49) hF8-(−573)-R5′-CATGCCCTTTCTCCTGACC-3′ A01 (SEQ ID NO: 50) hF8-(−683)-F5′-AGCAAGTGTTGAGGTCCAGG-3′ A02 (SEQ ID NO: 51) hF8-(−56)-R5′-TGAAGTAGCAAAAGGGAGGC-3′ A02 (SEQ ID NO: 52) hF8-(−168)-F5′-CTTCTCCATCCCTCTCCTCC-3′ A03 (SEQ ID NO: 53) hF8-441-R5′-CAGAAATGTTTCTTTGGGGC-3′ A03 (SEQ ID NO: 54) hF8-23012-F5′-CTTCAAATTTGCCTCCTTGC-3′ A04 (SEQ ID NO: 55) hF8-23436-R5′-AGACCAAGCAGAGGAAGACG-3′ A04 (SEQ ID NO: 56) hF8-25450-F5′-AATCTTGCCTCAGAGCAACC-3′ A05 (SEQ ID NO: 57) hF8-26066-R5′-GAAAAGCAATTCCTAGGGGG-3′ A05 (SEQ ID NO: 58) hF8-29413-F5′-GGGCAACAGAGTGAGACTCC-3′ A06 (SEQ ID NO: 59) hF8-30016-R5′-TTCTGGAACTCAGCTCCTCC-3′ A06 (SEQ ID NO: 60) hF8-35266-F5′-GGAGACCTGACATCAAAGCC-3′ A07 (SEQ ID NO: 61) hF8-35608-R5′-AACCCCATCTCCTTCATTCC-3′ A07 (SEQ ID NO: 62) hF8-37821-F5′-TAAGGTGTGAGCACACTGGG-3′ A08 (SEQ ID NO: 63) hF8-38404-R5′-CGATGAGTTCTGTTCTGAGCC-3′ A08 (SEQ ID NO: 64) hF8-52986-F5′-ATGGTGATTGGTGACCTTGG-3′ A09 (SEQ ID NO: 65) hF8-53544-R5′-GGAAACTAGGGGATCTTGGC-3′ A09 (SEQ ID NO: 66) hF8-55802-F5′-GTCTTGCTCCTGCTTTCACC-3′ A10 (SEQ ID NO: 67) hF8-56428-R5′-TACCCTTGCCATTTGATTCC-3′ A10 (SEQ ID NO: 68) hF8-56244-F5′-CTGCTGAAGAGGAGGACTGG-3′ A11 (SEQ ID NO: 69) hF8-56855-R5′-ATGTCCATTGGAGACAAGGC-3′ A11 (SEQ ID NO: 70) hF8-61345-F5′-GATTGTGGTATCTGCAGGGG-3′ A12 (SEQ ID NO: 71) hF8-61753-R5′-CAACAGCTGGAGAAAGGACC-3′ A12 (SEQ ID NO: 72) hF8-65333-F5′-TGACACTTTCACAGTCAACCG-3′ A13 (SEQ ID NO: 73) hF8-65920-R5′-CAGCAGGCACGTTTACTACG-3′ A13 (SEQ ID NO: 74) hF8-68456-F5′-CAGTCACCCTCTTGTCCTGG-3′ A14 (SEQ ID NO: 75) hF8-69067-R5′-GGGAATTAAAAGGGAGAGGG-3′ A14 (SEQ ID NO: 76) hF8-74699-F5′-CCTGGGAATAAGATAATGGGC-3′ A15 (SEQ ID NO: 77) hF8-75338-R5′-AAATGCTGGTGAGGATGTGG-3′ A15 (SEQ ID NO: 78) hF8-90867-F5′-ACAGCAGCAATGCAAAAACC-3′ A16 (SEQ ID NO: 79) hF8-91468-R5′-TCTATTGCTCCAGGTGATGG-3′ A16 (SEQ ID NO: 80) hF8-91365-F5′-ATGCTCTTGCGACAGAGTCC-3′ A17 (SEQ ID NO: 81) hF8-91942-R5′-AACAAAGCAGGTCCATGAGC-3′ A17 (SEQ ID NO: 82) hF8-91756-F5′-TTGGCAAAAAGTCATCTCCC-3′ A18 (SEQ ID NO: 83) hF8-92379-R5′-TAATTGCTTTGGACTGGGG-3′ A18 (SEQ ID NO: 84) hF8-92247-F5′-CCACCAGATGCACAAAATCC-3′ A19 (SEQ ID NO: 85) hF8-92850-R5′-TTTGCTTGGTTTGATTTCCC-3′ A19 (SEQ ID NO: 86) hF8-92700-F5′-GAAGGTTCATATGACGGGGC-3′ A20 (SEQ ID NO: 87) hF8-93290-R5′-ATGACTGCTTTCTTGGACCCC-3′ A20 (SEQ ID NO: 88) hF8-93200-F5′-TCTGACCAGGGTCCTATTCC-3′ A21 (SEQ ID NO: 89) hF8-93816-R5′-CATGATTGCTTTCACAAGCG-3′ A21 (SEQ ID NO: 90) hF8-93674-F5′-ATTGGATCCTCTTGCTTGGG-3′ A22 (SEQ ID NO: 91) hF8-94323-R5′-TGTCCCTGATTCCTCTACCC-3′ A22 (SEQ ID NO: 92) hF8-116050-F5′-ATGCAAAATGCTTCTCAGGC-3′ A23 (SEQ ID NO: 93) hF8-116647-R5′-AAAAGCTTGTTCAAAATAAATGG-3′ A23 (SEQ ID NO: 94) hF8-117559-F5′-TCTGTACCACTTCTTCCAGGG-3′ A24 (SEQ ID NO: 95) hF8-118125-R5′-TTTATGCCAGTCCAACCTGC-3′ A24 (SEQ ID NO: 96) hF8-118087-F5′-TATTTTTGGAAGGTGGGAGG-3′ A25 (SEQ ID NO: 97) hF8-118699-R5′-CGAATCCTTTGATCCTGAGC-3′ A25 (SEQ ID NO: 98) hF8-118318-F5′-TTGATGAGACCAAAAGCTGG-3′ A26 (SEQ ID NO: 99) hF8-118933-R5′-AGAGCATGGAGCTTGTCTGC-3′ A26 (SEQ ID NO: 100) hF8-120414-F5′-AAGCACTTTGCATTTGAGGG-3′ A27 (SEQ ID NO: 101) hF8-120947-R5′-TGGAGATCTTCGAGCTTTACC-3′ A27 (SEQ ID NO: 102) hF8-121066-F5′-GGACCCCAGTTTCTTCAGC-3′ A28 (SEQ ID NO: 103) hF8-121510-R5′-AGTGGGAAGTGGAGAGGAGG-3′ A28 (SEQ ID NO: 104) hF8-122636-F5′-GTATGCCATAAAGCCTTTATG-3′ A29 (SEQ ID NO: 105) hF8-123081-R5′-GTTCTAATCCCAGTAGAAGG-3′ A29 (SEQ ID NO: 106) hF8-126315-F5′-CCACAGCTTCACACACACAT-3′ A30 (SEQ ID NO: 107) hF8-126829-R5′-TCTATGAGCCTTGACACTAC-3′ A30 (SEQ ID NO: 108) hF8-159304-F5′-ttcccacttcttcttggtgc-3′ A32 (SEQ ID NO: 109) hF8-159934-R5′-tgggcatttaggttgactcc-3′ A32 (SEQ ID NO: 110) hF8-160633-F5′-tcatgccactacactccagc-3′ A33 (SEQ ID NO: 111) hF8-161150-R5′-ctgcccataaccaaacttcc-3′ A33 (SEQ ID NO: 112) hF8-161982-F5′-gggtgacagagcaagactcc-3′ A34 (SEQ ID NO: 113) hF8-162549-R5′-aaaaggcttgggaatcaagg-3′ A34 (SEQ ID NO: 114) hF8-184818-F5′-agatgtcccagatgcgtagg-3′ A35 (SEQ ID NO: 115) hF8-185411-R5′-GCTTTCATGCAGGTTTCTCC-3′ A35 (SEQ ID NO: 116) hF8-185329-F5′-TATTTTCTGCAGCTGCTCCC-3′ A36 (SEQ ID NO: 117) hF8-185941-R5′-CTTTCAACAATTGCATCCTCC-3′ A36 (SEQ ID NO: 118) hF8-185733-F5′-GAGGGGCACATTCTTATCTCC-3′ A37 (SEQ ID NO: 119) hF8-186382-R5′-TCATAGTGAAGGGGTCAGGC-3′ A37 (SEQ ID NO: 120) hF8-186289-F5′-CACCACACAATAGGATCCCC-3′ A38 (SEQ ID NO: 121) hF8-186832-R5′-GTCAATGGGAAAAGAATGCC-3′ A38 (SEQ ID NO: 122) hF8-186639-F5′-CAATCCACAAATGATGCAGG-3′ A39 (SEQ ID NO: 123) hF8-187259-R5′-AGTGCCAGGATTACAGGCAT-3′ A39 (SEQ ID NO: 124)

F. Examples

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how thecompounds, compositions, articles, devices and/or methods claimed hereinare made and evaluated, and are intended to be purely exemplary and arenot intended to limit the disclosure. Efforts have been made to ensureaccuracy with respect to numbers (e.g., amounts, temperature, etc.), butsome errors and deviations should be accounted for. Unless indicatedotherwise, parts are parts by weight, temperature is in ° C. or is atambient temperature, and pressure is at or near atmospheric.

1. Example 1

Recombinant factor VIII (r-fVIII) is the most widely-used and effectivetherapy for hemophilia A (hA). Many patients unfortunately becomerefractory when the wildtype (wt) protein is seen as foreign and istargeted by functionally neutralizing anti-fVIII antibodies termedinhibitors. While inhibitors occur most often in severe hemophiliacswith complex fVIII mutations and a complete circulating absence of thefVIII protein, recent studies show patients with missense-mutations(mMt) have a higher incidence than previously thought and demonstrater-fVIII can be immunologically targeted even when differing by only asingle amino acid. Because mMt represent the most frequent overalletiology of hA (−39% of cases), common non-hemophilic protein variantsof fVIII may represent an important novel modulator of inhibitordevelopment in this setting. To determine the extent of such variation,all coding regions of the fVIII loci (F8) were resequenced in 137healthy subjects from 7 ethnic groups, including 86 Caucasians and 16African-Americans (AA). Five common nonsynonymous single nucleotidepolymorphisms (nsSNPs) resulting in an amino acid change wereidentified. Whereas 4 were polymorphic in African-Americans (AA) only 2were variable in Caucasians, despite having examined 6× the number AAX-chromosomes. Recent studies show AA patients have a 2-fold higherinhibitor incidence than Caucasians, thus establishing ethnicity as arisk factor in this complication. Minor alleles for 2 nsSNPs arerestricted to AA and substitute amino acids in major B-Cell inhibitorepitopes located in the A2 and C2 domains. To confirm these findings andaccurately define the number and frequency of human haplotypes (H) (eg.distinct combinations in which the alleles of these 5 nsSNPs are linkedin vivo) F8 was resequenced in a second study group that included 75additional healthy AA. Here there are at least six distinct wt forms ofthe human fVIII protein by defining six haplotypes (H) from the fournsSNPs: H1, H2, H3, H4, H5, H6. H1 exists in all ethnic groups, is themost common overall form of fVIII and represents 2 of the 3 r-fVIIIconcentrates used clinically. While H2 is the most common form in AA(44%) and possibly represents the other therapeutic r-fVIII molecule, atleast 20% will have AA-restricted fVIII proteins; H4 (4%) and H5 (12%)Only 2 forms of fVIII were found in Caucasians, H1 (90%) and H2 (10%),in contrast, and neither were ethnically restricted. In summary, whencombined with reports of at least 2 other nsSNPs, it is established asinaccurate the long held view that fVIII is basically a monomorphicprotein in non-hemophiliacs. Greater immunologic barriers exist whenr-fVIII is infused into patients with mMt in endogenous FVIII moleculescontaining one or more minor alleles of these nsSNPs. Moreover, due tothe number and frequency of AA-restricted wt fVIII variants, thesensSNPs contribute pharmacogenetically to the higher incidence ofinhibitors in this ethnic group.

2. Example 2

A high-normal to elevated plasma FVIII activity (FVIII:C) level isassociated with increased risk for venous and arterial thrombosis.Despite being a highly heritable trait, the genes, other than the ABOblood group locus, responsible for the broad interindividual variance inFVBI:C remain to be identified. The structural FVIII locus (F8) isreported to have been excluded since earlier studies failed to detectlinkage or associations between FVIII:C levels and polymorphisms in ornear this candidate gene. However, because these studies were too smallto detect quantitative trait loci (QTLs) with modest effects or lowfrequencies, used genotyping methods less sensitive than sequencingand/or examined only portions of the gene, every known functional regionof F8 in 137 unrelated non-hemophilic individuals from different ethnicgroups was resequenced. 20 of the 46 single nucleotide polymorphisms(SNPs) identified were previously unknown despite their location in oneof the most intensely studied human genes. Because linkagedisequilibrium (LD) across F8 was weak overall, the relationship betweeneach SNP and FVIII:C was evaluated in 398 Caucasians from 21 Spanishfamilies—The Genetic Analysis of Idiopathic Thrombophilia Project(GAIT)—using marginal measured genotype association analysis. C92714G,which encodes the conservative B-domain substitution D1241E, wasassociated with significantly lower FVIII:C levels (each G-allelecontributes a 12.8 IU/dL reduction in activity) even after accountingfor important covariates, including age and ABO genotype. Nevertheless,studies in other populations and/or in vitro are necessary to determinewhether D1241E represents a functional QTL because the alleles ofG56010A, a SNP in the 3′ splice junction of intron 7, are in strong LDwith C92714G.

The (immature) large glycoprotein coagulation factor (F)VIII issynthesized as a single polypeptide chain containing 2351 amino acids.The immature polypeptide sequence for each haplotype is disclosed asfollows: H1 (SEQ ID NO: 13), H2 (SEQ ID NO: 14), H3 (SEQ ID NO: 15), H4(SEQ ID NO: 16), H5 (SEQ ID NO: 17), and H6 (SEQ ID NO: 18). Afterremoval of its 19 amino acid N-terminal signal-peptide in theendoplasmic reticulum, FVIII undergoes limited proteolysisintracellularly prior to secretion. The mature form of FVIII in plasmatherefore contains 2332 amino acids and is a heterodimer comprised of aconstant size light-chain and a non-covalently linked variable sizeheavy-chain. The mature polypeptide sequence for each haplotype isdisclosed as follows: H1 (SEQ ID NO: 19), H2 (SEQ ID NO: 20), H3 (SEQ IDNO: 21), H4 (SEQ ID NO: 22), H5 (SEQ ID NO: 23), and H6 (SEQ ID NO: 24).Heterodimeric FVIII is an inactive procofactor that circulates in anon-covalent, tightly bound complex with von Willebrand factor (VWF).Thrombin, generated in low concentrations at sites of vascular injury bythe extrinsic pathway during coagulation initiation, proteolyticallyconverts FVIII into its active, but labile, heterotrimeric cofactor form(FVIIIa), which is released from VWF. Upon assembly, FVIIIa participatesin the propagation phase of coagulation as a cofactor for thecatalytically active serine protease FIX (FIXa) within a macromolecularenzyme complex known as intrinsic Xase after binding to membranesurfaces containing anionic phospholipids, whose sole function is tocatalyze cleavage activation of its zymogen substrate FX. Hemophilia A,the X-linked bleeding disorder due to insufficient or absent FVIIIcoagulant activity (FVIII:C) in plasma, demonstrates that FVIII isindispensable for normal hemostasis. However a high-normal to elevatedplasma FVIII activity (FVIII:C) level is associated with an increasedrisk for both venous and arterial thrombosis.

Several environmental and endogenous factors are associated with broadinterindividual variability that exists for FVIII:C levels. Age, sex,oral contraception (OC), smoking, body mass index (BMI), diabetesmellitus (DM), ABO blood type, and plasma levels of total cholesterol(TC), low-density lipoprotein (LDL), triglyceride (TG), VWF, and FIXcoagulant activity (FIX:C) all have substantial evidence supportingtheir role as determinants of FVIII:C. The relative ease of identifying,observing, measuring and/or characterizing these factors enabledinvestigators to discern their relationships with plasma FVIII:C level.

The 186-kilobase (kb) F8 gene, which is located on the long-arm (q) ofthe X chr within Xq28.1, and consists of 26 exons that comprise onlyfive percent of the base pair (bp) total. Transcription may be robust togenetic variation in the promoter, particularly in the case of SNPswhich do not cause length changes that could markedly destabilize thepre-initiation complex whose DNA-binding proteins overlap thetranscription start site. The 1200-bp upstream DNA region from −1038 to+236, which contains most of the 5′-UTR, sufficiently promotedtranscription in several liver-derived cell lines. Furthermore, 300-bpof the immediately contiguous 5′-genomic DNA exerted maximal promoteractivity. This region contains several cis-elements that interact withDNA-binding proteins important for the transcriptional regulation ofthis gene; including a TATA-like sequence (GATAAA) located 30-bpupstream of the transcription start site. Additional cis-elements mayalso function as transcriptional regulators including a potentialrepressor located approximately 1-kilobase (kb) upstream of the gene.The presence of variants in this region of the promoter might contributeto “normal” variation of FVIII levels through variable transcriptionrates.

The F8 variation scan systematically covered the entire gene using alarge number of chromosomes to identify variations in. A scan of F8regions classically considered functional in 137 people for a total of222 X-chromosomes was performed. All of the 398 subjects of the GAIT(Genetic Analysis of Idiopathic Thrombophilia) project were genotypedfor the subset of variants polymorphic in the sample of Caucasians used.

F8 Reference Sequence

A 286-kb stretch of genomic DNA from Xq28.1 was obtained that containedthe 186-kb F8 sequence plus 50-kb of both 5′- and 3′-genomic sequencefrom the UCSC Genome Browser on Oct. 12, 2004, which corresponds to NCBIBuild 35 (hg17, the May 2004 release). This is the first version tocontain all 26 exons; specifically, it included exons 21 and 22, whichwere missing from previous releases. For purposes of referencing thegene and variants, the reverse compliment of this sequence was used andassigned +1 to the transcription start site (+1 GCTTAGTGCTGAG+13) thatand −1 to the base immediately 5′ to it. For instance, the promotercontains nucleotides with negative numbers and the putative TATA-box,GATAA, begins with G at base −30. “hg17” was used to indicate that thenucleotide numbering follows this convention.

Variaton Scan

The variation covered all exonic sequences (coding and untranslated),50- to 100-bp of intronic sequence from each splice junction,approximately 1-kb of the promoter, and about 300-bp of the immediatelyflanking 3′-genomic DNA. Table 1 lists the exonic regions, theirlengths, hg17 nucleotide positions, and the encoded amino acids, if any.DNA fragments were amplified and sequenced that were 500- to 600-bp inlength and, where necessary to cover an extended region, overlaped theprimers by approximately 100-bp. Initially, 39 amplicons were generated,but lacked coverage of exons 21 and 22. Though absent from the variationscan, amplicons (designated amp29B and amp30B) were included for thesetwo regions in the genotyping described below. Table 2 lists theamplicons and their hg17 nucleotide positions. Agencourt Biosciencesperformed the polymerase chain reaction (PCR) amplifications,cycle-sequencing reactions, and sequence determinations. The variationscan was performed using genomic DNA samples obtained from two discoverygroups of human subjects. The first wave included genomic DNAs from 90distinct individuals and one additional blind replicate. In addition,female GAIT founders were included (i.e., one unrelated female drawnfrom each of the GAIT families). The second wave consisted of 47 GAITfounders, 37 females and 10 males, with FVIII:C levels at the extremesof the founders. A total of 222 X-chromosomes were collected and to theamplicons were sequenced in the forward and reverse directions in bothof these waves.

TABLE 1 Characteristics of the 26 exons in F8. Exon Length (bp)Nucleotides* Amino Acids⁺ 01 (5′-UTR) 171  1 . . . 171 NA 01 (CDS) 143172 . . . 314  1 . . . 29 02 122 23124 . . . 23245 29 . . . 70 03 12325629 . . . 25751  70 . . . 111 04 213 29576 . . . 29788 111 . . . 18205 69 35419 . . . 35487 182 . . . 205 06 117 37921 . . . 38037 205 . . .244 07 222 53172 . . . 53393 244 . . . 318 08 262 56037 . . . 56298 318. . . 405 09 172 56583 . . . 56754 405 . . . 462 10 94 61556 . . . 61649463 . . . 494 11 215 65553 . . . 65767 494 . . . 565 12 151 68682 . . .68832 566 . . . 616 13 210 74817 . . . 76026 616 . . . 686 14 3106 91048. . . 94153  686 . . . 1721 15 154 116151 . . . 116304 1721 . . . 177216 213 117701 . . . 117913 1773 . . . 1843 17 229 118200 . . . 1184281844 . . . 1920 18 183 118636 . . . 118818 1920 . . . 1981 19 117 120557. . . 120673 1981 . . . 2020 20 72 121282 . . . 121353 2020 . . . 204421 86 122773 . . . 122858 2044 . . . 2072 22 156 126492 . . . 1266472073 . . . 2124 23 145 159497 . . . 159641 2125 . . . 2173 24 149 160858. . . 161006 2173 . . . 2222 25 177 162116 . . . 162292 2223 . . . 228126 (CDS) 153 184972 . . . 185124 2282 . . . 2332 26 (3′-UTR) 1806 185125. . . 186930 NA *Nucleotide numbering corresponds to the hg17 referencesequence for F8. ⁺Amino acids whose codons are completely or partlycontained in each exon; numbering based on the mature protein found inplasma (after removal of the 19 residue signal peptide), whoseN-terminal alanine (amino acid # 1) corresponds to the20^(th)-translated residue; NA—not applicable.

Genotyping

TABLE 2 F8 regions sequenced to identify and genotype commonpolymorphisms. Amplicon* Forward Primer Reverse Primer Nucleotides⁺ 015′-TTATCAAAGGGGCTTCTTGC 5′-CATGCCCTTTCTCCTGACC  −1214 . . . −573 025′-AGCAAGTGTTGAGGICCAGG 5′-TGAAGTAGCAAAAGGGAGGC   −683 . . . −56 035′-CTTCTCCATCCCTCTCCTCC 5′-CAGAAATGTTTCTTTGGGGC   −168 . . . 441 045′-CTTCAAATTTGCCTCCTTGC 5′-AGACCAAGCAGAGGAAGACG  23012 . . . 23436 055′-AATCTTGCCTCAGAGCAACC 5′-GAAAAGCAATTCCTAGGGGG  25450 . . . 26066 065′-GGGCAACAGAGTGAGACTCC 5′-TTCTGGAACTCAGCTCCTCC  29413 . . . 30016 075′-GGAGACCTGACATCAAAGCC 5′-AACCCCATCTCCTTCATTCC  35266 . . . 35608 085′-TAAGGTGTGAGCACACTGGG 5′-CGATGAGTTCTGTTCTGAGCC  37821 . . . 38404 095′-ATGGTGATTGGTGACCTTGG 5′-GGAAACTAGGGGATCTTGGC  52986 . . . 53544 105′-GTCTTGCTCCTGCTTTCACC 5′-TACCCTTGCCATTTGATTCC  55802 . . . 56428 115′-CTGCTGAAGAGGAGGACTGG 5′-ATGTCCATTGGAGACAAGGC  56244 . . . 56855 125′-GATTGTGGTATCTGCAGGGG 5′-CAACAGCTGGAGAAAGGACC  61345 . . . 61753 135′-TGACACTTTCACAGTCAACCG 5′-CAGCAGGCACGTTTACTACG  65333 . . . 65920 145′-CAGTCACCCTCTTGTCCTGG 5′-GGGAATTAAAAGGGAGAGGG  66456 . . . 69067 155′-CCTGGGAATAAGATAATGGGC 5′-AAATGCTGGTGAGGATGTGG  74699 . . . 75338 165′-ACAGCAGCAATGCAAAAACC 5′-TCTATTGCTCCAGGTGATGG  90867 . . . 91468 175′-ATGCTCTTGCGACAGAGTCC 5′-AACAAAGCAGGTCCATGAGC  91365 . . . 91942 185′-TTGGCAAAAAGTCATCTCCC 5′-CTAATTGCTTTGGACTGGGG  91756 . . . 92379 195′-CCACCAGATGCACAAAATCC 5′-TTTGCTTGGTTTGATTTCCC  92247 . . . 92850 205′-GAAGGTTCATATGAGGGGGC 5′-ATGACTGCTTTCTTGGACCC  92700 . . . 93290 215′-TCTGACCAGGGTCCTATTCC 5′-CATGATTGCTTTCACAAGCG  93200 . . . 93816 225′-ATTGGATCCTCTTGCTTGGG 5′-TGTCCCTGATTCCTCTACCC  93674 . . . 94323 235′-ATGCAAAATGCTTCTCAGGC 5′-AAAAGCTTGTTCAAAATAAATGG 116050 . . . 11664724 5′-TCTGTACCACTTCTTCCAGGG 5′-TTTATGCCAGTCCAACCTGC 117559 . . . 11812525 5′-TATTTTTGGAAGGTGGGAGG 5′-CGAATCCTTTGATCCTGAGC 118087 . . . 11869926 5′-TTGATGAGACCAAAAGCTGG 5′-AGAGCATGGAGCTTGTCTGC 118318 . . . 11893327 5′-AAGCACTTTGCATTTGAGGG 5′-TGGAGATCTTCGAGCTTTACC 120414 . . . 12094728 5′-GGACCCCAGTTTCTTCAGC 5′-AGTGGGAAGTGGAGAGGAGG 121066 . . . 12151029B 5′-GAATTTAATCTCTGATTTCTCTAC 5′-GAGTGAATGTGATACATTTCCC 122740 . . .122902 30B 5′-TAAAAATAGGTTAAAATAAAGTG 5′-TTTAAATGACTAATTACATACCA 126453. . . 126668 29A 5′-TCAGGGTTGGTTACTGGAGC 5′-ACACTACCATGGTCTTGGGG 158245. . . 158735 30A 5′-AGTCAGTGGGCCTGTTATGG 5′-GTCCCTAGCTCTTGTTCCCC 158526. . . 159105 31 5′-TGGGCAGATAGGGATAGTGG 5′-TTTGTGCGTTTCTCAACAGC 158833 .. . 159409 32 5′-TTCCCACTTCTTCTTGGTGC 5′-TGGGCATTTAGGTTGACTCC 159304 . .. 159934 33 5′-TCATGCCACTACACTCCAGC 5′-CTGCCCATAACCAAACTTCC 160633 . . .161150 34 5′-GGGTGACAGAGCAAGACTCC 5′-AAAAGGCTTGGGAATCAAGG 161982 . . .162549 35 5′-AGATGTCCCAGATGCGTAGG 5′-GCTTTCATGCAGGTTTCTCC 184818 . . .185411 36 5′-TATTTTCTGCAGCTGCTCCC 5′-CTTTCAACAATTGCATCCTCC 185329 . . .185941 37 5′-GAGGGGCACATTCTTATCTCC 5′-TCATAGTGAAGGGGTCAGGC 185733 . . .186382 38 5′-CACCACACAATAGGATCCCC 5′-GTCAATGGGAAAAGAATGCC 186289 . . .186832 39 5′-CAATCCACAAATGATGCAGG 5′-AGTGCCAGGATTACAGGCAT 186639 . . .187259 *To include all known functional F8 regions in the variationscan, 41 distinct segments (i.e., amplicons) of this structural locuswere PCR amplified from genomic DNA samples and resequenced directly. Togenotype the 12 F8 variations that were polymorphic in Caucasians (Table3) in the entire GAIT cohort the 11 amplicons in bold were generated bythe PCR and resequenced directly. ⁺Nucleotide numbers correspond to thehg17 reference sequence for F8.

Chromatograms were used to both detect and genotype variants at allstages. Agencourt used the PHRAP suite (www.phrap.org) to identifyvariants. PHRED output was used and a custom written SAS_(—)8.2 programwas used to genotype individuals at the variant bases reported byAgencourt. In addition, the SAS program generated a separatechromatogram for each variant that placed the base of interest betweentwo 10-bp flanks. By being compact and centering on the variant, thesechromatograms standardized the manual review of the calls, whennecessary. In the remaining GAIT individuals not in the variation scan,Agencourt sequenced the subset of amplicons in which at least onevariable nucleotide was found among the Caucasians in the forwarddirection only. Finally, to verify the accuracy of the genotyping, asmeasured by the ability to confirm the original genotyping call, asubset of samples was replicated. When necessary to resolve any missinggenotypes, amplicons were generated and sequenced. When it was necessaryto perform manual reviews, forward and reverse sequences were pairedfrom the same individuals, but the file names were not un-blinded, whichrevealed the well address and amplicon. INTER (PEDSYS, San Antonio,Tex.) was used to search for violations of Mendelian inheritance withinthe GAIT project, blanking any resulting discrepancies.

Statistical Analyses

The program Genecounting was used to estimate r² and D′, measures ofallelic association. The program SOLAR was used to create plots of thesestatistics.

The measured genotype approach was employed to determine whether, amongthe members of the GAIT project, there were marginal genotype-specificdifferences in the mean FVIII:C level, adjusted for relevantenvironmental and endogenous factors. To account for thenon-independence among family members, the analyses was performed usingthe program SOLAR, a likelihood-based variance component geneticanalysis program. Any FVIII:C value was set beyond four standarddeviations from the mean to missing.

Data was had for the following covariates: age, sex, smoking status, OCuse, ABO blood group genotype, total cholesterol (TC), high densitylipoprotein (BDL), low density lipoprotein (LDL), very low densitylipoprotein (VLDL), triglycerides (TG), lipoprotein(a), BMI, diabetesmellitus (DM) status, VWF:Ag, and FIX:C. ABO genotypes were representedwith indicator variables. For instance, AO had a value of one if thepatient's genotype was AO, and it had a value of zero otherwise. A zeroin each of the variables (AA, AB, AO, and BO) indicated the OO genotype,the reference level. Both smoking status and OC use were representedwith dichotomous variables that indicated any versus no use, thereference level. Simple analyses were performed for each of the abovevariables to evaluate the association with FVIII:C levels. In thesefirst analyses the covariate was the sole independent variable except inthe case of ABO, which consisted of indicator variables, and age, whichalso had higher order terms. Further, bivariate analyses were performedwith the endogenous factors and FVIII:C levels to investigate a sharedgenetic source. Those factors were excluded that shared commonunderlying genetic influences with FVIII:C from the final measuredgenotype analysis.

Because SNPs, including single-base-substitutions (SBSs) andinsertion/deletions (INDELs), have a major (M) and minor (m) allele,there are five possible genotypes for each. Specifically, there arethree genotypes in females who have two X-chromosomes (homozygousM-allele [X_(M)|X_(M)], heterozygous [X_(M)|X_(m)] and homozygousm-allele [X_(m)|X_(m)]), and two in males who receive their mother'sX-chromosome and father's Y-chromosome (hemizygous M-allele [X_(m)|Y]and hemizygous m-allele [X_(m)|Y]. Thus, the genotype of each SNP wasrepresented with a value of either 1 ([X_(M)|X_(M)] and [X_(M)|Y]), 0([X_(M)|X_(m)]), or −1 ([X_(m)|X_(m)] and [X_(m)|Y]), respectively,since no a priori knowledge was had of which allele might be associatedwith increased FVIII:C levels. Initial complex analyses were performedseparately for each SNP in which age, age², sex, age×sex, age²×sex, AA,AB, AO, BO, smoking status and OC use were covariates to test fordifferences in the marginal genotype-specific mean FVIII:C levels.Subsequently analyses were performed using all of the availablecovariates previously mentioned for any SNP suggestively associated withFVIII:C levels at the p=0.10 level. The reason behind this tactic was tonot enforce the same mechanistic effect on every SNP, but to allow forpotential individual actions. In addition, age was represented byemploying linear splines with knots at 15 and 50 years. A logarithmictransformation of FVIII:C levels was used to improve normality and aconstant multiplier of 8.2 to avoid potential precision problems withnumeric iteration in the statistical routines.

FVIII:C Levels

The mean±standard deviation (SD) for the FVIII:C levels in the GAITstudy subjects was 150.7 IU/dL (±52.2 IU/dL), after excluding two womenwhose FVIII:C levels were more than 4 SD from the mean. FIG. 7 presentsthe FVIII:C levels versus age indicating the sex of each subject.

Variation Scan

46 distinct SNPs were identified, which included 45 SBS and one INDEL,among the 19,157-bp covered in the variation scan. Table 3 lists thesevariants, their protein and genic details, and whether the UW-FHCRC scanor dbSNP also reported them. In this table, bold print indicates thevariants genotyped in the GAIT subjects and the italics indicatenonsynonymous SNPs.

TABLE 3 F8 Polymorphisms F8 Variation Amino Minor Allele Frequencies*Databases^(a) Nucleotide Acid Total* C AA Ch SEA J M SAA HAMD UW- Gene(position/ (position/ (n = (n = (n = (n = (n = (n = (n = (n = (HAM-FHCRC NCBI (region) alleles) alleles) 222) 144) 26) 13) 15) 8) 8) 8)STeRS) (VDR) (dbSNP) Promoter G-000825A 0.9% NP 7.7% NP NP NP NP NP NFF8-002289 NF Promoter G-000824A 0.9% NP 7.7% NP NP NP NP NP NF F8-002290NF Promoter G-000493A 0.5% NP 3.8% NP NP NP NP NP NF F8-002621 rs4898404Promoter 4A: -000385: 5a 0.9% NP NP NP NP NP 25.0% NP NF NF NF PromoterT-000287C 0.5% NP 3.8% NP NP NP NP NP + NF NF Intron 02 G025610A 0.5% NP4.5% NP NP NP NP NP + F8-028722 NF Intron 03 G025865A 0.5% NP 4.2% NP NPNP NP NP NF F8-028977 NF Intron 03 G025885C 0.5% NP 4.2% NP NP NP NP NPNF F8-028997 NF Intron 03 C029567T 0.5% NP NP NP NP NP NP 12.5% NF NF NFIntron 04 T029854C 0.5% NP 3.8% NP NP NP NP NP NF NF NF Intron 05C035518G 0.9% NP 8.0% NP NP NP NP NP NF NF NF Intron 06† A053034G 0.5%0.7% NP NP NP NP NP NP NF NF NF V. Exon G053206T VI. 0.5% NP NP NP  6.7%NP NP NP  +* NF NF W0255C Intron 07† C055938A 0.5% NP NP NP  6.7% NP NPNP NF NF NF Intron 07† G056010A 9.6% 6.4% 26.9%  7.7% NP 12.5% 12.5%25.0% + F8-059122 rs7058826 Exon 08† G056113A A0343A 0.5% NP 3.8% NP NPNP NP NP + F8-059225 rs1800289 Intron 09 T061534C 0.5% NP 3.8% NP NP NPNP NP NF F8-064646 rs5986899 Exon 10 G061620A H0484R 0.9% NP 8.3% NP NPNP NP NP + NF NF Intron 13 A090948G 0.5% NP NP NP NP NP NP 12.5% NF NFNF Exon 14 A091317G R0776G 0.5% NP NP 7.7% NP NP NP NP NF NF rs2228152Exon 14† C092555T H1188H 0.5% NP 4.3% NP NP NP NP NP + F8-095667 NFC092714G D1241E 14.7%  7.7% 72.7%  7.7% NP 12.5% 12.5% 25.0% + F8-095826rs1800291 A092798C S1269S 7.7% 6.3% 3.8% 7.7% 20.0% NP 12.5% 25.0% +F8-095910 rs1800292 Exon 14 G092927A K1312K 0.5% NP NP NP  6.7% NP NP NPNF NF NF Exon 14 C093401G V1470V 0.5% NP 4.5% NP NP NP NP NP NF NF NFExon 14 G093434A P1481P 0.9% NP 9.1% NP NP NP NP NP + NF NF Intron 15C116434T 0.5% NP NP 7.7% NP NP NP NP NF NF NF Intron 18† T118909A 20.3% 14.6%  84.6%  7.7% 14.3% NP 42.9% 37.5% + F8-122021 rs4898352 Intron 19†T120776C 24.5%  17.3%  75.0%  7.7% 18.7% NP 37.5% 50.0% + F8-123888rs4074307 Intron 22 C158352T 0.5% 0.7% NP NP NP NP NP NP NF NF NF Intron22 T158368C 0.5% 0.7% NP NP NP NP NP NP NF F8-161500 NF Intron 22C158635T 0.5% NP 3.8% NP NP NP NP NP + F8-161767 rs5987054 Intron 22A156777G 0.5% 0.7% NP NP NP NP NP NP NF NF NF Intron 22 C158820T 0.5% NP3.8% NP NP NP NP NP + F8-161952 rs5987053 Intron 22 G159087A 0.5% 0.7%NP NP NP NP NP NP NF NF NF Intron 23† 1.4% 1.4% 3.8% NP NP NP NP NP NFF8-163006 NF Intron 24† G162013T 3.3% 5.0% NP NP NP NP NP NP + F8-165145NF Exon 25† A162161G M2238V 1.8% NP 15.4%  NP NP NP NP NP NF F8-165293rs1705196 Intron 25† T162475C 4.1% 5.6% NP NP NP NP NP 12.5% + F8-165607NF 3′-UTR† C185156T 0.6% NP 3.8% NP NP NP NP NP NF F8-188288 rs59868873′-UTR† G186341A 0.5% NP NP NP NP 12.5% NP NP NF NF NF 3′-UTR† A186506G0.5% 0.7% NP NP NP NP NP NP NF NF NF 3′-UTR† 0.5% 0.7% NP NP NP NP NP NPNF NF NF 3′-UTR† A186799G 24.8%  17.4%  76.9%  NP 13.3% NP 50.0% 50.0% +F8-189931 rs1050705 3′-gDNA† T186987G 0.9% NP 9.1% NP NP NP NP NP NFF8-190119 NF 3′-gDNA† T187064C 1.8% NP 15.4%  NP NP NP NP NP NFF8-190196 NF *Frequencies estimated in 137 unrelated subjects fromdifferent ethnic groups: Caucasians (C), both Caucasian-American andSpanish-Caucasian; African- American (AA); Chinese (Ch); Japanese (J);Southeast Asian, excluding Ch and J (SEA); Mexican Indian (MI); andSouth American Andes (SAA). Genotype data were not complete for allsubjects and variants, resulting in denominators that varied from themaximum number of X chromosomes for a group. Underlined SNPs were theones genotyped in the entire GAIT cohort. †The bolded coding region SNPsemphasize the non-synonymous subset. ^(a)Databases with F8polymorphisms: HAMD—the HAMSTeRS Hemophilia-A Mutation Database (+indicates a previously known polymorphism; *denotes missense mutationcausing mild HA); VDR—the Variation Discovery Resource (denotes variantsby gene and nucleotide position; located at University ofWashington-Fred Hutchinson Cancer Research Center [UW-FHCRC]);dbSNP—database for Single Nucleotide Polymorphisms (denotes variant witha unique identifier; located at the NCBI). NF—not found.

Genotype data were not complete for all subjects and variants, resultingin denominators that varied from the maximum number of X chromosomes fora group. Underlined SNPs were the ones genotyped in the entire GAITcohort. ^(†) The bolded coding region SNPs emphasize the non-synonymoussubset.

^(a)Databases with F8 polymorphisms: HAND—the HAMSTeRS Hemophilia-AMutation Database (+ indicates a previously known polymorphism; *denotes missense mutation causing mild HA); VDR—the Variation DiscoveryResource (denotes variants by gene and nucleotide position; located atUniversity of Washington-Fred Hutchinson Cancer Research Center[UW-FHCRC]); dbSNP—database for Single Nucleotide Polymorphisms (denotesvariant with a unique identifier; located at the NCBI). NF—not found.

Most of the variants had very low minor allele frequencies (MAF) and, itfollows, were present in only one of the racial groups. For instance, 21SNPs were present only in the group of subjects designated as AfricanAmerican. Of these, only two, A 162161G and T187064C, had MAF above 10%.Only eight SNPs were present in more than one group, two of which werepresent in only two groups (Table 4). For these six alleles, the MAF hadgreat range within the groups such that the description minor allele isa misnomer. For example, the minor allele at nucleotide C92714G inCaucasians was G (7.7%), but with an estimated frequency of 72.7% inAfrican Americans, it is the major allele in this ethnic group.

TABLE 4 Covariate Beta Estimate p-Value Age (years) −0.132 0.0167 Sex0.253 0.0209 Age × Sex 9.377 × 10⁻⁰⁴ 0.9914 A_15* 0.126 0.0289 A_15 ×Sex 0.015 0.8693 A_50* 0.074 <0.0001 A_50 × Sex −0.061 0.0066 OralContraceptive Use (Yes/No) −0.632 0.0271 Smoking Status (Yes/No) −0.2880.0121 AA 0.882 <0.0001 AO 0.648 <0.0001 AB 0.727 0.0553 BO 0.668 0.0019BMI (kg/m²) 0.132 0.3439 Triglyceride (mM) 0.362 0.0084 D1241E (+1, 0,−1) 0.312 0.0244 *Coefficient for the splines with knots at 15 and 50years.

Allelic-Association

FIG. 8 presents the estimated r² and D′ between each pair of alleles,respectively. (FIG. 8A) The hg17 sequence used as a reference for F8 inthis study spans about 287-kb and contains the approximately 187-kbstructural locus and 50-kb of both the 5′ and 3′ flanking genomic DNA.The F8 locus consists of 26 exons (triangles) and 25 introns(intervening black lines). The 1.2-kb and 0.3-kb promoter and flanking3′-genomic segments scanned for variations are indicated. The numerousrepetitive elements (RepeatMasker) in this gene are indicated bydifferent colored triangles. (FIG. 8B) The 41 amplicons used forvariation detection and genotyping are indicated by open boxes. (FIG.8C) The 46 SNPs identified in the variation scan, and two additionalSNPs (G75215A and T186594C) discovered by genotyping the extended GAITcohort for the polymorphism subset variable in at least Caucasians, aredesignated by their gene location (left) and nucleotide position in thetranscription unit. Promoter SNPs are located upstream of −1. Major- andminor-alleles are shown on the left and right. The 17 exonic- and 11coding-region-SNPs are also indicated by their nucleotide and amino acidpositions in the polyadenylated mRNA and mature form of the circulatingFVIII protein, respectively. Finally, the five coding region SNPsresulting in nonsynonymous amino acid substitutions are shown in green;G53206T is actually a missense mutation identified in a non-affectedfemale carrier since Trp255Cys represents a known cause of mildhemophilia A in Chinese individuals.

Measured-Genotype Analysis

Age, smoking status, OC use, DM status, BM and the plasma levels of TC,LDL, VLDL, TG, VWF and FIX:C were associated with FVIII:C levels at thep=0.10 level. In addition, sex and its interactions with age wereincluded, despite non-significant results in the univariate analysis.Bivariate analyses of FVIII:C levels with both FIX:C and VWF levelssuggested that they shared common underlying genetic influences and thuswere excluded from the multiple covariate measured-genotype analysis.

Only the analyses for G56010A, an intron 7 SNP located 27 nucleotidesupstream of its 3′-splice junction (i.e., position −27), and thenon-synonymous coding region SNP C92714G, which encodes the knownaspartate to glutamate substitution in the B-domain residue 1241,suggested significant differences in the genotype-specific mean FVIII:Clevels. Therefore, these SNPs were further explored separately in a morecomplex model. Table 4 presents the results of the final model in theanalysis of D1241E and FVIII:C levels. Among the GAIT founders who hadno parental data, the D′ between these two SNPs was 0.84. Since theresults were similar, more complete genotyping data was had for D1241E(one DD female, 2 DE females, and 3 EY males had missing genotypes forG56010A), and D1241E results in an amino acid change, the results forthis SNP were presented.

When LDL and DM status were included in the full model, thebeta-estimate for the effect of D1241E was 0.295 (p=0.0356). Excludingthese variables did not meaningfully affect the point estimate orimprove the precision (Table 4), suggesting that these factors were notconfounding the relationship between this SNP and FVIII:C levels. WhenVLDL was substituted for TG levels in the above model, the resultingestimate of the effect of D1241E was 0.300 (p=0.0294). As expected,Pearson's correlation coefficient between TG levels and VLDL was high,p=0.96 (p<0.0001), which did not account for non-independence betweenthese observations. The estimate of the correlation coefficient wassimilar when the analysis was restricted to (independent) founders.

In the completed a variation scan of the known functional regions of theF8 using 222 X-chr from unrelated, non-hemophilic patients two variantswere found whose alleles were highly associated with each other, G56010Aand C92714G (corresponding to amino acid D1241E), and also significantlyassociated with FVIII:C levels. Interestingly, there was not strongassociation between the alleles (see FIG. 9) of F8 variants overall. Asfar as C92714G is concerned, only four polymorphisms had alleles with aD′ of approximately 0.50: G56010A, A118909T, C120776T, and A186799G.Among the GAIT members that had no parental data (i.e., founders), theD′ between these alleles and C92714G were 0.84, 0.46, 0.47, and 0.54,respectively.

FIG. 9 illustrates the pattern and degree of LD across F8 in varioushuman populations. The subset of 47 F8 polymorphisms variable in the 148unrelated Caucasian subjects studied were evaluated pairwise for LD.Both commonly used parameters of LD (i.e., D′ and r²) were determinedusing maximum likelihood haplotype frequencies estimated by theexpectation-maximization (EM) method implemented in GENECOUNTING.Individual polymorphisms are listed along the y-axis in their 5′ to 3′gene orientation and along the x-axis according to their nucleotideposition within the F8 genomic sequence. The estimates of LD based on D′are shown above the pair-wise line of identity, which passes through theorigin, while those based on r² are shown below. The color legend onright illustrates semi-quantitatively the possible degrees of LD thatcan exist between the alleles of any polymorphism pair; white (LD=0)indicates linkage equilibrium while red (LD=1) indicates complete LD orallelic association.

The distribution of FVIII:C levels, even within members of the 21Spanish families of the GAIT project, displayed the typical variabilityof this trait and had an estimated heritability 0.40. Several factorswere controlled for in the final analysis. The support for theassociation between age and FVIII:C levels is among the strongest. Thegraph of crude FVIII:C levels versus age appears and the results of theanalysis also supports this relationship (see FIG. 7 and Table 4,respectively). The use of splines with age allowed for a less rigorousimposition on the data, namely that the same fit not be enforced acrossthe entire range of approximately 2 to 87 years. For example,flexibility was allowed in the effects of age on FVIII:C levels in theyoung and the old, instead of modeling, for instance, a quadrant effectacross both periods in which the age data for the old influences theestimated response for the young, and vice versa.

The results of the analysis support the finding that smokers had lowermean FVIII:C levels than non-smokers. As detailed information on smokingwas lacking, this relationship was not further elucidated in theanalyses.

OC use had an apparently large effect, to that for ABO blood group (seeTable 4).

There is also strong support for an association between FVIII:C levelsand ABO blood group and between FVIII:C levels and VWF. In separatebivariate analyses with FVIII:C levels, it was found that each of VWFand FIX had potential overlap in the set of (unknown) genes that mayaffect their trait levels. Since variance components-based analyses wereperformed as implemented in the program SOLAR, in which the covariancebetween family members is used to estimate the additive geneticvariance, including such variables in the analyses was not germane tothis example. Further, the inclusion ABO blood type, which has apotential pleiotropic effect on VWF and FVIIIC levels, also supports theexclusion of VWF

Removal of TC/LDL from the model had negligible impact upon the pointestimate of the effect of the SNP and its precision. Removal of TG/VLDLhad a noticeable impact and VLDL was included. The choice between TG andVLDL was arbitrary and the results were very similar.

1-81. (canceled)
 82. A purified FVIII having a haplotype selected fromthe group consisting of the H3 haplotype, H4 haplotype, H5 haplotype,and H6 haplotype.
 83. The FVIII of claim 82 having the H4 haplotype. 84.The FVIII of claim 82 having the H5 haplotype.
 85. The FVIII of claim 82having the H6 haplotype. 86-92. (canceled)
 93. The FVIII of claim 82having the H3 haplotype.
 94. The FVIII of claim 82 with instructions foradministration to a person who has been haplotyped with the samehaplotype as the recombinant FVIII.
 95. The FVIII of claim 82 having theH3 haplotype with instructions for administration to an African Americanhemophiliac.
 96. The FVIII of claim 82 having the H4 haplotype withinstructions for administration to an African American hemophiliac. 97.The FVIII of claim 82 having the H5 haplotype with instructions foradministration to an African American hemophiliac.
 98. The FVIII ofclaim 82 having the H6 haplotype with instructions for administration toa non African American hemophiliac.
 99. The FVIII of claim 82 having theH6 haplotype with instructions for administration to a Chinesehemophiliac.
 100. A method for treating a hemophiliac comprisingadministering to the hemophiliac an effective amount of purified FVIIIhaving a haplotype selected from the group consisting of the H3haplotype, H4 haplotype, H5 haplotype, and H6 haplotype.
 101. The methodof claim 100, wherein the recombinant FVIII has the H3 haplotype. 102.The method of claim 100, wherein the FVIII has the H4 haplotype. 103.The method of claim 100, wherein the recombinant FVIII has the H5haplotype.
 104. The method of claim 100, wherein the FVIII has the H6haplotype.